Method for selecting a single cell expressing a heterogeneous combination of antibodies

ABSTRACT

The present invention provides combinations of specific binding proteins, such as immunoglobulins, that are designed to be true combinations, essentially all components of the combination being functional and compatible with each other. The invention further provides a method for producing a composition comprising at least two different proteinaceous molecules comprising paired variable regions, the at least two proteinaceous molecules having different binding specificities, comprising paired variable regions, at least two proteinaceous molecules having different binding specificities, comprising contacting at least three different variable regions under conditions allowing for pairing of variable regions and harvesting essentially all proteinaceous molecules having binding specificities resulting from the pairing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT International PatentApplication No. PCT/NL2004/000386, filed on May 28, 2004, designatingthe United States of America, and published in English, as PCTInternational Publication No. WO 2004/106375 A1 on Dec. 9, 2004, whichapplication claims priority to European Patent Application No.03076671.1 filed on May 30, 2003, the contents of the entirety of eachof which is incorporated herein by this reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Pursuant to 37 C.F.R. §1.52(e)(5), the sequence listing has beensubmitted by CD-R, and is hereby incorporated by reference in itsentirety. The CD-Rs are labeled “Copy 1” and “Copy 2”, respectively, andeach disc contains one file entitled “2183-7585us seq list.txt” which is47 KB and was created on Mar. 24, 2006.

TECHNICAL FIELD

The present invention relates to the field of molecular biology, inparticular, to medical molecular biology.

BACKGROUND

Specific recognition plays an important role in modern medical biology.Receptor-ligand interactions, immune responses, infections, enzymaticconversions are all based on specific recognition between molecules. Ofparticular interest are specific protein-protein interactions, whichgive a vast array of possibilities to interfere in all kinds ofbiological processes. Throughout nature, biological processes are foundthat depend on more than one (simultaneous) protein-interaction. At thepresent time, it seems that interfering at more than one point in abiological process is going to be more effective than a singleinterference. Particularly in antibody therapy, it is seen that one(monoclonal) antibody is often not effective enough for treating aparticular disorder and/or disease. Therefore, the attention of manymedical researchers is now focused on combination therapies. Well-knownexamples of combinations of antibodies that are presently clinicallypursued are for the treatment of non-Hodgkin's lymphoma, the combinationof the already approved anti-CD20 antibody Rituxan with the anti-CD22antibody Epratuzumab from AmGen, and for the treatment of Hepatitis B, acombination of two human antibodies being developed by XTLPharmaceuticals (E. Galun et al., Hepatology (2002) 35:673-679).However, the combination of multiple (two or more) drugs (be itantibodies or other) has a number of technical, practical and regulatorydrawbacks. The drugs were typically not designed as combinations anddevelopment with optimal clinical efficacy and compatibility may be aproblem. As an example, conditions for stabilizing the one may bedetrimental to stability of the other(s). Furthermore, multiple sourcesof recombinant production lead to multiple sources of risks, such as,viral contamination, prion contamination and the like.

The present invention provides combinations of specific bindingproteins, such as immunoglobulins, that are designed to be truecombinations, essentially all components of the combination beingfunctional and compatible with each other. By producing truecombinations, the present inventors have opened up an avenue of furtherimprovements in both the production and properties of the combinations.These improvements and their advantages will become apparent from thefollowing description.

SUMMARY OF THE INVENTION

Thus, the invention provides a method for producing a compositioncomprising at least two different proteinaceous molecules comprisingpaired variable regions, the at least two proteinaceous molecules havingdifferent binding specificities, comprising contacting at least threedifferent variable regions under conditions allowing for pairing ofvariable regions and harvesting essentially all proteinaceous moleculeshaving binding specificities resulting from the pairing. Bindingspecificities are defined as interactions between molecules that can bedistinguished from background interactions. Typically, specificinteractions between molecules have higher binding affinity thanbackground interactions between molecules.

Specific binding molecules, which for an important part are made up ofamino acid residues (proteinaceous molecules), often require the pairingof different amino acid sequences in order to build a binding site. Anamino acid sequence that pairs with another amino acid sequence to builda binding site is referred to as a variable region herein. Of course,such a sequence may be part of a larger amino acid sequence, which mayagain be part of a larger proteinaceous molecule, e.g., as a subunit. Asan example, in an antibody a complementarity-determining region (CDR)may be a variable region, but a combination of three CDRs with theirframework regions may also be considered as a variable region. Accordingto the present invention, at least two different binding sites are builtin one system, in one method. Thus, variable regions (amino acidsequences) are brought together under conditions in which they may pairto build two different binding sites. This requires at least threevariable regions, of which one is capable of pairing with both othervariable regions, thus building two specific binding sites. The twospecific binding sites may be in one proteinaceous molecule or indifferent proteinaceous molecules, or both.

In antibodies of the IgG isotype, for example, this would be an antibodyhaving two identical or two different binding sites. By producing thetwo desired binding specificities in one system, there is only onesource of the products and thereby less risk of contamination withviruses, prions and the like. Such a system may be a cell-free system,such as a wheat germ system, but it is preferred to carry out methodsaccording to the invention inside a cell, or more cells of the sameorigin, preferably the origin of the subjects to be treated, typicallyhuman. For production and selection purposes, other cells, such as,bacteria, insect cells, yeasts and other eukaryotes may typically bepreferred.

If the pairing of the variable regions takes place in a cell, then it ispreferred that the production of the variable regions also takes placein a cell, preferably the same cell. A particularly useful way ofproducing variable regions is through the expression of nucleic acidsencoding these variable regions. It is preferred that all variableregions in one cell are produced by such expression, it is, however,also possible to produce a number of variable regions in this manner andhave other variable regions brought in, based on different techniques ofproduction, or the same means of production, but in another cell. Formost purposes, the nature of the nucleic acid is not critical, it may beRNA, is preferably DNA, may be episomal or integrated, part of a viralvector or a plasmid, etc. However, for the final production system ofthe combination of proteins having different binding specificities, itis preferred that the nucleic acid or acids encoding the variableregions are stably integrated into the host genome. Production ofvariable regions through expression of nucleic acids encoding them givesthe possibility to manipulate the encoding sequences, thereby enablingthe designing of new binding specificities, better pairing properties,exchanging useful sequences from one encoding sequence to another andthe like. It also gives the possibility for selection for improved ordifferent binding and/or pairing properties after alterations have beenmade, giving rise to the creation of libraries of many different nucleicacids in systems with easy selection mechanisms.

In this manner, the number of variable regions to be expressed forobtaining different binding sites may be reduced. One may design and/orselect for a so-called promiscuous variable region, which is capable ofpairing with more than one different binding region. “Pairing” isdefined herein as any kind of coming together to build a binding site,be it through covalent or noncovalent bonding, conformationalarrangement, folding, dimerization, multimerization or any other way. Itthus encompasses terms such as associating, assembling, binding,combining and the like, be it directly or indirectly. Particularly whenmore than two different binding specificities are made in one cell, itis useful to have promiscuous variable regions in such a system,reducing the number of different nucleic acids that have to beexpressed. In such a system, the promiscuous variable region should notcontribute significantly to the binding specificity of the pairedregions. Preferably, it is mostly involved in folding and stability ofthe binding site, thereby, of course, indirectly influencing the bindingspecificity.

Apart from reducing the number of nucleic acids to be expressed, bychoosing one or more promiscuous variable regions, the number of pairedvariable regions which are not functional can be reduced to essentiallyzero.

Particularly in the field of immunoglobulins, which typically comprisetwo pairs of two different paired variable regions, the production ofmore than one immunoglobulin inside the same cell often leads to pairingof variable regions that does not lead to a desired binding specificity.In the present invention, pairs are designed such that in one systemessentially all variable regions can pair with another in the system toform a useful specific binding site. In methods of the prior art whereinfour variable regions were expressed in hybrid-hybridomas or quadromas,the result was a low percentage of desired bispecific antibodies, apercentage of either original antibodies and a substantial percentage ofpaired regions without significant useful binding specificity.Bispecific antibodies may be produced with the methods according to thepresent invention, either together with or without the concomitantproduction of the original antibodies, but typically essentially withoutproduction of non-functional pairs. In addition, mixtures of multiplemonoclonal and multiple bispecific antibodies may be produced with themethods according to the present invention.

The methods as disclosed in the detailed description provide foradaptation of the nucleic acids encoding variable regions to the desiredend result. Using promiscuous pairing or the opposite, monogamouspairing, the end result can be designed. Where bispecific antibodies orother certain pairings are to be excluded, the use of pairs of variableregions that can pair only with each other is used. Further, methods asdisclosed in the detailed description provide for adaptation of thenucleic acids encoding the constant regions to lead to a preferentialpairing of the binding sites formed by the variable regions whenattached to the constant regions.

Antibodies in the present invention are intended to refer to allvariations of immunoglobulins that retain specific binding, such asFabs, Fab′2, scFvs, but typical for antibodies according to theinvention is the presence of a pair of amino acid sequences (at leasttwo CDRs) that are paired to form a binding site. Thus, the inventionalso provides a method wherein the variable regions are derived fromheavy chains and/or light chains of immunoglobulins, engineered versionsof variable regions with elements of heavy and/or light, chains ofimmunoglobulins and/or a method wherein the proteinaceous molecules areantibodies, fragments and/or derivatives of antibodies.

The methods according to the invention are typically preferred for theproduction of multiple (i.e., three or more) binding specificities inone system. Because of the specific design of the contributing variableregions this has now become technically and commercially feasible.

Another element of the invention useful for control of the production isplacing expression of different variable regions under control ofdifferent elements such as promoters, (trans) activators, enhancers,terminators, anti-repressors, repressors, and the like. These controlelements may be inducible or repressible. Thus, the production ofvariable regions can be regulated, thus optimizing pairing conditions asdesired. Different combinations of variable regions can be made byseparation in time of expression of various variable regions and/orratios between different paired variable regions may be manipulated byregulating expression levels. Variations are described in the detaileddescription. The invention also provides an expression system forcarrying out a method according to the invention, comprising nucleicacids encoding variable regions together with all elements required forgene expression and pairing, preferably such an expression systemcomprises at least one recombinant cell, such as a bacterium, a yeastcell, a fungal cell, an insect cell, a plant cell or another eukaryoticcell, in particular, a mammalian cell, more in particular, a human cell.

Such a system can be provided with all necessary and useful controlelements as disclosed herein before and as well known in the art.Selection elements and suicide elements may also be introduced into sucha system as desired.

A collection of expression systems according to the invention comprisinga variety of combinations of different specificities is also provided,typically as a library for use in selecting desired combinations ofvariable regions.

Such selection methods are also part of the present invention. Thus, theinvention in one embodiment also provides a method for selectingcombinations of proteinaceous molecules having specific affinity for atleast two target epitopes, comprising contacting a collection accordingto the invention with the two target epitopes and selecting combinationsshowing the specific affinity.

Such methods are particularly useful when the two target epitopes areassociated with one disease or disorder. It is preferred to combine sucha method with subjecting a selected combination of proteinaceousmolecules to a biological assay indicative of an effect of thecombination on the disease and/or disorder.

Compositions obtainable by the methods of the invention are also part ofthe present invention. Preferred are compositions comprising at leastthree different paired variable regions, having different bindingspecificities, in particular, those wherein the variable regions arederived from immunoglobulin light chains and/or immunoglobulin heavychains. A combination composition that targets both TNF-α as well asIL-1β is an exemplary combination of the invention. In such typicaltherapeutic uses it is important that the combination preparations donot lead to severe immune responses in the subject to be treated. Atleast some of the antigenic parts of the binding molecules, such as theconstant regions in antibodies should be of human origin. In thealternative, antigenic parts may be omitted or masked by molecules suchas PEG. Thus, the invention also provides in one embodiment acomposition according to the invention, which is a pharmaceuticalcomposition. Although antibodies have found use in other areas, andantibody combinations according to the present invention can be used inother areas, the pharmaceutical use of the invented combinations ispreferred, both diagnostic and therapeutic, with a preference for thelatter. However, in industrial applications the combinations of theinvention may also be superior to existing separation techniques,because of ease of production, consistency of production and theavailability of many combinations of specificities, capable ofseparating almost anything from any mixture. In testing, be it inpharmaceutical diagnostics or in any other field (environmental,agricultural, to name a few) the combinations of the invention can beused advantageously as well. Both partners of a sandwich assay can bemade in one cell. Agglutination mixtures can be made in one cell. Whenusing the IgG format, the expression in the same cell will lead to asubstantial fraction of bispecific compounds, which offer uniqueapplications in combination with the monoclonals present in the samemix. For example, when a monoclonal antibody can only bind with one armto an antigen, a bispecific molecule with binding sites capable ofbinding to two different epitopes on the same antigen, may moreconsistently than the monoclonal antibody mixture immobilize or trapantigen. Again, ease and consistency of production, as well as thediversity of specificities is an asset of the combinations of theinvention. These advantages of course also apply in selecting andproducing combinations of specificities for therapeutic and/orprophylactic use, with additional advantages in ease of selection,efficacy of selected combinations and the mentioned safety aspects.

A simple combination according to the invention starts with twospecificities present in the combination. When a promiscuous variableregion is present, such a combination requires only three differentvariable regions. The combination can be made such that all resultingpaired variable regions in one proteinaceous molecule have the samespecificity, giving monospecific molecules, or the variable or, ifappropriate, the constant regions can be designed such that bispecificmolecules are also present. It can also be designed such that onemonospecific and one bispecific molecule are present, but that the otherpossible monospecific molecule does not arise, because the variableregions cannot assemble in that manner. Thus, the invention in oneembodiment comprises a composition comprising at least one monospecificantibody and at least one bispecific antibody produced in one cell foruse as a pharmaceutical. In some applications bispecific molecules,especially antibodies, may be advantageous for bringing two antigenstogether on a cell surface. Such aggregation events are often requiredin biology for transduction of a signal to the inside of a cell.Bispecific antibodies in the mixture may also be used to connecteffector molecules with target cells. The uses envisaged for bispecificantibodies in the prior art are also envisaged for bispecific moleculesaccording to the invention. The most advantageous compositions accordingto the invention comprise more than two different monospecific bindingmolecules, optionally together with the different possible combinationsof bispecific or multispecific molecules that may result from thedifferent possible pairing events. These multispecific mixtures resemblepolyclonal mixtures in their efficacy for recognizing antigens, butwithout the drawbacks of many irrelevant specificities in the mixture.The mixtures resemble monoclonal antibodies in their definedconstitution, ease of production and high specificities, but without theconcomitant loss of efficacy. The mixtures according to the inventionare referred to as Oligoclonics™. Oligoclonics™ can thus contain two,three, or more different binding specificities, and can exist in variousformats. In the simplest form, Oligoclonics™ in the IgG format contain amixture of different monospecific antibodies and bispecific antibodiesin a particular given ratio. In the Fab format, Oligoclonics™ contain amixture of different Fab molecules which are the product of correctlypaired variable regions. In the mixed format, Oligoclonics™ contain amixture of antibodies and antibody fragments.

As disclosed herein, the methods and means of the invention in oneembodiment are the production of combinations of specificities. Beforeproduction of combinations, suitable combinations must be designedand/or selected. These methods for designing and selection are also partof the present invention. Thus, in a further embodiment, the inventionprovides a method for producing nucleic acids encoding variable regionsfor use in a method for production of combinations of specificitiesaccording to the invention comprising synthesizing nucleic acidsencoding variable regions, expressing the nucleic acids and allowing theexpression products to pair and selecting nucleic acids encodingvariable regions having desired pairing behavior.

In an alternative embodiment, the invention provides a method forproducing nucleic acids encoding variable regions for use in a methodfor production of combinations of specificities according to theinvention comprising altering existing nucleic acids encoding variableregions, expressing the nucleic acids and allowing the expressionproducts to pair and selecting nucleic acids encoding variable regionshaving desired pairing behavior. Of course, both methods may be combinedand/or repeated in any order. Synthesis, alteration and selectionmethods are disclosed in more detail in the detailed description.

Preferred nucleic acids (also part of the invention) for use inproducing combinations of specificities are those encodingimmunoglobulin polypeptides. Of course all types of immunoglobulins,especially antibodies (IgM, IgE, IgGs, etc.) but also fragments (scFv,Fab, single-domain, engineered variants) can be used in the presentinvention. Variable regions can, for example, be derived from eitherimmunoglobulin heavy chain variable regions, or immunoglobulin lightchain variable regions, but can also be engineered hybrids of heavy andlight chain variable regions (with, for example, swapped CDR regions orFR regions). Variable regions can, for example, be obtained fromhybridomas, by cloning from immune or non-immune donors or can besynthetically constructed variable regions. Even hybrids can be producedusing nucleic acids and methods of the invention. For example, hybridswith different yet functional binding sites can be made by providingelements from different isotypes, for example, IgM and IgG, or IgM andIgA. It should be born in mind that T cell receptors, resembleantibodies in many respects. Thus, the methods according to theinvention can also be applied advantageously with T cell receptors,their variable regions and their encoding nucleic acids. It is thuspreferred that the invention is carried out using immunoglobulins havingdifferent chains (T cell receptors), especially antibodies having lightchains and/or heavy chains or parts/derivatives thereof. Of course partand/or derivatives according to this invention are such parts and/orderivatives that do have specific binding properties comparable toimmunoglobulins.

This means that variable regions according to the invention should atleast comprise an element which resembles a complementarity-determiningregion of an antibody (CDR). Preferably it should have more than a CDR,preferably a variable region resembles in size and physicochemicalproperties a VH or VL of an antibody. The detailed description describesthe invention using antibodies as an exemplary embodiment of theinvention.

The invention will be described in more detail in the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Examples of composition of three or six proteinaceous moleculeswith three different binding specificities. The use of antibodies withappropriate pairing between the variable regions yields mixtures ofantibodies that are bispecific or monospecific and bivalent (top panel).By appropriate engineering to manipulate the pairing between thevariable regions, mixtures of only bispecifics or bivalent moleculesarise (left hand side panels). In the legend on the right panel (greybox) it is indicated that the three symbols, the circle, triangle andsquare, represent binding sites consisting each of variable regions.

FIG. 2: Method to identify antibodies with pairing-compatible elementsby empirical analysis of antibody variable region combinations.

FIG. 3: Antibodies with similar light or heavy chain by selection fromlibraries with restricted diversity. In this example of a Fab library,one of the antibody chains is identical in all library members (thewhite chain), while the others contain amino acid diversity.

FIG. 4: Different approaches to select antibodies with appropriatepairing behavior. (a) selection of Fab library with constant lightchain, and equivalent for Fab library with diversity in light chain onlyin (d); (b) selection of antigen-binding single-domain antibody fromheavy chain only library, and equivalent for VL in (e); (c) selection oflibrary of chimeric chains of VH and VL (in which, for example, some CDRelements are swapped).

FIG. 5: Selecting antibodies with pairing-compatible variable regions byre-shuffling one chain. Starting point of the method is a repertoire ofantibody binding sites, with paired variable regions, such as in thisexample, an Fab repertoire. Similarly single chain Fv libraries can beused. In a typical selection (top) the initially present pairing ofvariable regions is maintained throughout the iterative selectionprocess; in the selection followed by reshuffling (steps 1-3), one ofthe two variable regions (preferably of the heavy chain) of the pairsthat have been selected on antigen, is combined with partner domains(preferably light chains) derived from either the selected population orfrom the original population). After this the selection (step 4), andthe subsequent procedure are repeated. Eventually, individualantigen-reactive antibodies are identified by screening methods.

FIG. 6: Example of a competitive selection of antibodies with adesirable pairing behavior. The method involves the co-expression of oneor more competing antibodies (top, left) in the same host cell as amember of an antibody library (bottom, left). Depicted is the method forFab fragments, as described in the text. The result of the pairingopportunities of VHCH1 (white boxes) chain when co-expressed with twoother Fab fragments is depicted. The original combination of the VH withits cognate light chain (hatched box), will retain its original bindingaffinity for antigen and can thus be selected.

FIG. 7: Identifying antigen-specific antibodies by co-transfecting heavychain gene libraries with an invariant light chain gene and screeningthe resulting antibody mixtures for antigen reactive antibodies. Withevery cycle of transfection and screening, the diversity of the VHlibrary is reduced (at position *), to eventually yield a population ofantigen-reactive heavy chain variable genes. The numbers indicate thatsampling of a library of 10⁸ different heavy chains can be carried outby screening the wells of ten 96-well tissue culture with each 100clones per well.

FIG. 8: Identifying antigen-specific antibodies by transfectingsecretable heavy chain gene libraries, assembly with an invariant lightchain and screening the resulting antibody mixtures for antigen reactiveantibodies. With every cycle of transfection and screening, thediversity of the VH library is reduced (at position *), to eventuallyyield a population of antigen-reactive heavy chain variable genes.

FIG. 9: Screening antibody mixtures produced by the same host cell foroptimal bio-activity. Mixtures are made by transfecting heavy chaingenes encoding the antibodies of interest (here number is 10) togetherwith optimally paired light chain, followed by cloning of cell lines,selecting stably producing cell lines, and eventually screening theresulting antibody mixtures for optimal bio-activity.

FIG. 10: Examples of antibodies with cross-over domains. Heavy chaindomains (grey striped boxes) and light chain domains (white boxes).

FIG. 11: Ex vivo assembly of antibodies (A) and the universal antibodyconcept (B). Antibodies are produced as separate chains and thencombined to form a functional antibody. This is in particularlyinteresting when making mixtures of antibodies, as indicated in (B),where depending on the input of the chains and the separation of themixing reactions.

FIG. 12: Dependent expression of Ig chains. Chain-1 is typically theheavy chain, which is under control of a promoter (P). The IRES sequencelinks the expression of the heavy chain with that of a transactivator;this activates a responsive promoter to induce expression of Chain-2,typically the light chain (see text for details).

FIG. 13: The sequence of pSCFV (SEQ ID NO:46), a pUC119-based plasmidsuitable for stepwise cloning of antibody variable regions andexpression of scFv fragments.

FIG. 14: Schematic depiction of plasmid pSCFV-3 (A) and pSCFV-3 withthree cloned scFv fragments, in this case derived from the antibodiesJA, JB and M57. The black box is a schematic depiction of the histidinestretch; other C-terminal-based tags are also indicated. S, signalsequence; rbs, ribosome binding site; AMPr, ampicillin resistance gene(beta-lactamase).

FIG. 15: Schematic depiction of the eukaryotic expression vectorVHExpress as also described in (Persic et al. (1997) 187:9-18) exceptthat this variant has a CMV promoter; its use for cloning scFv fragments(top, indicated for antibody JA) such that the expression of scFv-Fcfusions is achieved.

FIG. 16: Sequence alignment of the three light chains amino acidsequences of antibodies JA (Kappa) (SEQ ID NO:8), and JB (SEQ ID NO:10)and M57 (SEQ ID NO:12) (both lambdas). The position of the CDRs isindicated.

FIG. 17: pFAB-display: Schematic depiction of pFAb-display (top), andindication of cloning of VLCL and VH regions; the polylinker region(below). Legend as in FIG. 14.

FIG. 18: Mutagenesis of heavy chain variable region of the JA antibody(SEQ ID NO:7); underlined region was mutagenized. Other regions known tobe important for the interaction with the VL: the residues at thepositions marked in color (bottom) or with the boxes around the JA-VHsequence are, alternatively, suitable for mutagenesis (based on datafromworldwideweb.biochem.unizh.ch/antibody/Structures/DimContacts/VHDimHistFrame.html).

FIG. 19: Outline of an expression vector for human monoclonal antibodiesin eukaryotic cells. CMV: CMV promoter; p(A): polyadenylation signal;Neo: neomycin resistance gene; Amp: ampicillin resistance gene.

FIG. 20: Outline of the expression cassette and expression vectors foruse with eukaryotic cells. The legend of the vector elements is depictedon the right. On the left hand side top panel are depicted, as examples,four eukaryotic expression cassettes for three antibody heavy chains andone light chain. The elements found in an expression cassette for asingle antibody chain encoding gene or nucleic acid typically comprisesa promoter, a Leader sequence, an open reading frame encoding theantibody chain of interest, a polyadenylation region and terminator, allin operable configuration. Further sites/regions used for site-directedand in some cases homologous, recombination, are shown (are alsooptional; indicated on top of the first expression cassette). On thebottom panel is depicted an exemplary vector backbone used for insertionof the top panel cassette(s). This scheme displays the typical elementsof a eukaryotic expression vector, comprising a bacterial origin ofreplication (such as Col E1), a bacterial selection marker (B-Select,such as the ampicillin resistance gene), a eukaryotic selection marker(Select, such as gpt, neo, zeo, etc., see text; useful when stableintegration into the host cell's genome is envisaged), and additionaloptional elements such as a bacteriophage packaging region (for ss-DNAproduction, such as f1), and an optional amplification marker (such asDHFR). Optional are other expression controlling elements (such as BEs,STAR, LCRs, MARs and the like, see below) and IRES; these are includedin later figures.

FIG. 21: Schemes depicting different formats for the co-expression ofantibody chain encoding genes, exemplified here for the case in whichtwo antibodies that share a common light chain (not shown) have to beco-expressed. (A) The basic individual cassettes, as separate cassettesand cloned into separate expression vectors. (B) This cassette containsthe two Heavy chain (H) genes cloned in tandem, but their expression isindividually regulated, via two different promoters, P1 and P2. (C) Thetwo H genes are cloned into transcriptionally opposite directions and inthis example separated by an element that influences theexpression/stability/integration frequency (further examples are givenin the text). (D) Same as B, but now additional E-elements are includedat the 3′ end of each of the two transcriptional units. (E) For cases inwhich two binding proteins should be present in the mixture at roughlysimilar quantities, an IRES is inserted between two H genes. (F and G)Expression cassettes for mediating the expression of two H chains, inwhich each of the H genes are linked via an IRES element to a selectionmarker (which is then selected for instead of using thevector-backbone-based marker), without (G) or with (H) additionalelements in one cassette to influence expression.

FIG. 22: Plasmid pABExpress40 for expression of libraries ofpairing-compatible antibodies in mammalian cells. Cloning sites fordirectional insertion of antibody variable region genes are indicated.See Example 11 for details. Without the STAR40 insertion into the EcoRIsite, this plasmid is called pABExpress.

FIG. 23: Design of a hybrid light chain library for identifying apairing-compatible light chain for h4D5v8 and 2C4. The amino acidsequences used by Herceptin (trastuzumab, h4D5v8) and pertuzumab(Omnitarg, 2C4) are compared to one another, and to two designer lightchain libraries, HYB1 and HYB2 (see Example 17 for details of thedesign). Residues identical to those of Herceptin are indicated with adash; amino acids are encoded by the single-letter consensus; X meanspositions to be targeted for diversification in a library approach.Numbers indicated for the most relevant residue positions (see text formore details).

FIG. 24: Plasmid p2Fab-HER2 used for the identification of a light chainvariable region that is pairing-compatible with two HER2-bindingantibodies, h4D5v8, and 2C4. The black box is a schematic depiction ofthe histidine tag (six Histidines); other C-terminal-based tags are alsoindicated. S, signal sequence; rbs, ribosome binding site; AMP^(r),ampicillin resistance gene (beta-lactamase). The version of the VL ofh4D5 that is present in this vector carries two designed mutations intwo CDR residues, and a stop codon (indicated with *) in the CDR2region. By site-directed mutagenesis, the CDR2 is diversified using anoligonucleotide (designed according to approach HYB2) thatsimultaneously removes the stop codon as well as introduces diversity atthree positions of the CDR2. This plasmid directs the expression of twoantibody heavy chains (as Fd chains) and one antibody light chain, andthus allows simultaneous production, and individual detection, of twoFab fragments.

FIG. 25: Growth inhibition curves for h4D5 Fab and mixtures of 4D5* and2C4* (see Example 17) that utilize different light chains, indicatedwith VL1 to VL7. Different concentrations of these Fabs are incubatedwith HER2-positive cells sensitive to the growth inhibitory effect ofHER2-targeting antibodies.

DETAILED DESCRIPTION OF THE INVENTION

In the fight against infection, the immune system creates a cellular andhumoral response that can specifically combat the infectious agent. Thehumoral immune response is based on immunoglobulins, or antibodies,which contact antigens and mediate certain effector functions to clearthe infection ((I. M. Roit, et al. (1985)) and all references herein).In the immune system antibodies are generated by B-lymphocytes.Antibodies consist of heavy and light chains that are assembled viainter-domain pairing and interchain disulphide bonds to form multivalentmolecules. Various isotypes of natural antibodies exist, including IgG(within humans, four subclasses, IgG1, IgG2, IgG3, IgG4), IgM, IgD, IgAand IgE. An IgG molecule contains two heavy (H) and two light (L)chains, both with a variable (V) and constant (C) regions. A typical IgGantibody comprises two heavy (H) chain variable regions (abbreviatedherein as VH), and two light (L) chain variable regions (abbreviatedherein as VL). The VH and VL regions can be further subdivided intoregions of hypervariability, termed “complementarity-determiningregions” (“CDR”), interspersed with regions that are more conserved,termed “framework regions” (FR). The extent of the framework region andCDRs has been precisely defined (see, E. A. Kabat, et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242,and C. Chothia, et al. (1987) J. Mol. Biol. 196:901-917, which areincorporated herein by reference).

In the generation of the primary immune response, the pairing of heavyand light variable region sequences of antibodies is a random process.The variable region genes are first assembled by recombining of a setrandomly picked V. (D) and J genetic elements represented in the genomeas a diverse gene pool. The recombined heavy and light variable regionsare then spliced towards their respective constant region genes and thechains expressed, assembled and secreted as immunoglobulin. In thiscombinatorial library, in principle every heavy chain can pair withevery light chain, to create a vast repertoire of different antigenspecificities, with diversity derived from the rearrangement process(which also introduces further diversity at some of the segmentjunctions) and from the combinatorial assembly of the heavy and lightchain variable regions. In principle, B-cells produce only one antibodyspecificity, encoded by one antibody heavy and one antibody light chainsequence. The immune system selects via an efficient antigen-selectionprocess those antibodies that can bind to a given antigen, inparticular, when the antigen is foreign and part of a pathogen.

In natural immunoglobulins, the light chain which consists of twodomains, is paired to the heavy chain, which consists of at least fourdomains and a hinge region: non-covalent interactions occur between VHand VL, and between CH1 and CL; between the latter a disulphide bridgeprovides a covalent linkage between heavy and light chains. Furthermore,the heavy chains are found paired to one another, i.e., in the IgGformat, and sometimes further associate with additional elements such asJ-chains (i.e., in the IgM format). A strong non-covalent interactionoccurs between the CL and CH1 domains, a frequently weaker interactionis present between VL and VH. The heavy chains are paired viainteractions in the hinge region (often covalently associated via one ormore disulphide bridges) and between the CH2 and CH3 domains. Bysequencing large pools of antibody variable genes from isolated B-celland comparing the frequency of the pairings of VH and VL segments, itwas confirmed that this pairing between VH and VL regions is on averagea random process. However, since the variable regions are geneticallydiverse and some of this diversity at the amino acid level isstructurally situated at the predicted interface region between the twodomains, the pairing of one given VH to another VL is not any morerandom. For example, pairing of a given VH with another VL than themolecule was initially selected with, may lead to loss of affinity ofbinding for the antigen, but may also lead to a reduced pairingefficiency. Within one B-cell, typically and normally only one light andone heavy chain is expressed, but in the few instances that other lightor heavy chains are expressed (such as in two fused B-cells), mispairingbetween the chains will occur, and antigen binding is lost in thisfraction of the antibody preparation. For example, in the past, theexpression of multiple antibody variable domains, as in quadromas orcells transfected with multiple heavy and/or light chain genes,typically yields a large fraction of pairings of variable regions thatare not functional. In order to build bispecific antibodies, the pairingof different antibody heavy and light chains when expressed in the samecell was investigated intensively. From studies of the pairing inantibodies derived from hybrid hybridomas made by fusing twoantibody-producing hybridomas, the pairing was shown to be based on arandom association of light and heavy chains with some cases where acertain level of preferential pairing was seen, but not enough toprevent mispairing to occur.

The present invention describes a variety of methods to selectantibodies with optimal pairing behavior of antibody chains. With suchmethods compositions of multiple antibodies with different bindingspecificities can be made.

1. Antibodies with Pairing-Compatible Variable Regions

a. Summary

Herein, we disclose methods and means for obtaining antibodies withpairing-compatible variable regions. The presence of such variableregions facilitate the predictability and functionality of the resultingpairing between the antibody variable regions. Two antibodies containpairing-compatible variable regions when the pairing of the variableregions in a mixture of all variable regions combined, occurs in suchmanner that predominantly functional binding sites arise as a result ofthe pairing. Two antibodies have pairing-compatible variable regionswhen, for example, the variable light chain domains of both antibodiescan be exchanged by the one of the other antibody, without drasticallyaltering the antigen-binding affinity of the two antibodies. Anotherexample of when antibodies have pairing-compatible variable regions, iswhen they share an identical or closely related variable region. In thatcase, pairing of the two partner domains to this shared region will leadto the formation of functional binding sites.

Methods for the identification of antibodies that havepairing-compatible variable regions are described. In the simplest formpairing-compatible variable regions in sets of antibodies are identifiedby virtue of the sequence identity of the V-regions. In another approachpairing-compatible variable regions are identified by empirical exchangeof V-genes or V-gene fragments between given antibodies, and testingantigen binding. In another approach, antibodies with a high likelihoodof containing pairing-compatible variable regions can be enriched fromantibody repertoires by combinations of selections and re-shuffling.Using appropriate selection strategies, antibody pairing may be selectedto become promiscuous or exclusive in the context of the desiredmultiple antibody variable genes. A method is also described forproviding a given antibody with pairing-compatible variable sequencing,using various mutagenesis and selection technologies. In anotherapproach, antibodies with pairing-compatible variable regions areselected from synthetic antibody libraries with a high probability ofidentifying antibodies with such elements (for example, from a librarywith only one variegated variable domain). Further, antibodies withpairing-compatible variable regions are created by first selecting anantigen-specific single-domain antibody, and then providing this with asecond domain that will pair with the first one to form a two-domainmolecule.

Pairing-compatible variable regions can be identified in order toreplace sequences in an antibody by the equivalent sequences of anotherantibody that are thought to mediate more favorable characteristics. Thetransfer of pairing-compatible variable regions between antibodies canbe used to alter the pairing capability and pairing strength of theantibody chains, but it can also be envisaged to alter theimmunogenicity, idiotype and expression yield of antibodies. Antibodiesbearing such elements are also highly suitable for making pharmaceuticalcompositions of antibodies with multiple binding sites, for example, formaking mixtures of antibodies containing such elements, by co-expressionin the same host cell. In particular, when the variable regions share afull variable domain (such as the light chain), co-expression will yieldfunctional binding sites only. Antibodies with pairing-compatiblevariable regions are suitable for the creation of mixtures ofantibodies, in which the antibodies are either solely monospecific, orbispecific, or a mixture of mono- and bispecific antibodies, or even,depending on the choice of isotypes with more than two binding sites(e.g., sIgA, IgM), combinations of multiple specificities within thesame antibody molecule. Such approaches provide a means to have in thesame pharmaceutical preparation antibodies with multiple specificities,and, if required, combinations of specificities within the samemolecule.

b. Sources of Antibodies

Antibodies suitable for the invention can be derived from a variety ofsources, including monoclonal antibodies, phage antibodies, antibodiesfrom transgenic animals etc. Monoclonal antibodies are obtained from apopulation of substantially homogeneous antibodies using the hybridomamethod first described by Kohler and Milstein, Nature 256:495 (1975) ormay be made by recombinant DNA methods. In the hybridoma method, a mouseor other appropriate host animal, is immunized to elicit lymphocytesthat are capable of producing antibodies that will specifically bind tothe antigen used for immunization. Alternatively, lymphocytes may beimmunized in vitro. Lymphocytes are fused with myeloma cells using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. Antibodies can also be isolated from transgenic animals thatharbor human immunoglobulin genes.

Antibodies or antibody fragments can also be isolated usingdisplay-based antibody library technology, wherein antibody fragmentsare selected by exposing a library of such antibodies displayed on thesurface of phage, yeast or other host cell, to the antigen of interest,and isolating those antibody fragments which bind to the antigenpreparation. A display library is a collection of entities; each entityincludes an accessible polypeptide component and a recoverable componentthat encodes or identifies the peptide component. Many antibodyfragments have been displayed on the surface of entities that carry thegenetic material encoding the antibody fragment inside the entity, suchas bacteriophages. This format is termed “phage display.” Phage displayis described, for example, in Ladner et al., U.S. Pat. No. 5,223,409;Smith (1985) Science 228:1315-1317. Other display formats utilizepeptide-nucleic acid fusions. Polypeptide-nucleic acid fusions can begenerated by the in vitro translation of mRNA that includes a covalentlyattached puromycin group, e.g., as described in Roberts and Szostak(1997) Proc. Natl. Acad. Sci. U.S.A. 94:12297-12302, and U.S. Pat. No.6,207,446. The mRNA can then be reverse transcribed into DNA andcross-linked to the polypeptide. In still another display format thelibrary is a cell-display library. Proteins are displayed on the surfaceof a cell, e.g., a eukaryotic or prokaryotic cell. Exemplary prokaryoticcells include E. coli cells, B. subtilis cells, spores, exemplaryeukaryotic cells include yeast such as Saccharomyces cerevisiae,Hansenula polymorpha, Pichia pastoris, Kluyveromyces lactis, insectcells and mammalian cells. Methods for the display of antibody fragmentsand the construction of antibody libraries in a variety of formats arewell described in the literature and known to those skilled in the art.

c. Identifying Pairing-Compatible Elements in Panels of Antigen-ReactiveAntibodies

Antibodies with pairing-compatible variable region sequences and,therefore, suitable pairing behavior of variable regions, are identifiedby a variety of methods that are disclosed within this document. In afirst approach, antibodies with pairing-compatible variable regions areselected from panels of antigen-specific antibodies (in which theantigen can be one defined target antigen but also a collection ofdifferent antigens, and the panel contains at least two antibodies), asfollows. The sequences of heavy and light variable regions aredetermined and inspected to find clones with identical or highly similarlight or heavy chain variable domains. If the amino acid sequence ofpart of or the complete variable region is identical for two antibodies,the two given antibodies have a pairing-compatible variable region.

In another approach, pairing-compatible variable regions are identifiedin amino acid sequences that appear related yet have amino aciddifferences: for example, if there are differences in the amino acidsequence but the same or related germ line segment is used, or whenhighly similar CDR regions are used, or if similar canonical folds insome CDR regions are found yet different germ line segments are used,the variable regions may still comprise pairing-compatible variableregions. This is confirmed by swapping the variable region(s) betweenthe antibodies in the panel, and measuring antigen binding of the newpairs. Experimentally light and heavy chains or parts thereof can beexchanged by recombinant DNA methods such as restriction enzyme-basedDNA cloning, oligonucleotide-based mutagenesis, gene synthesis andPCR-mediated mutagenesis, methods which are widely available in the art.Binding assays that can be used are well established in the art andknown to those skilled in the art; some are described below. This methodmay identify cases in which both variable regions can be exchangedbetween two antibodies, such as two related light chains that can beswapped with no or an acceptable effect on the affinity. It can alsoidentify cases in which only one of the variable regions of the twoantibodies can tolerate the exchange, for example, one light chain thatfunctionally pairs with one of two heavy chains only, while the otherlight chain can functionally pair with both heavy chains. In that casethe latter light chain can be used to replace the former non-matchingone and, thus, create two antibodies with pairing-compatible variableregions. Functional pairing means that the variable region pairing hasideally no effect on antigen-binding affinity or specificity, butallowable may also be a <10-fold reduction in affinity, and at the mosta 100-fold reduction in affinity, or any improvement of affinity.

In another embodiment, pairing-compatible variable regions areidentified in panels of antibodies without knowing or using the sequenceof the variable regions of the antibodies. First a collection ofantibody variants is created in which all variable regions are combinedwith the other partner variable regions of the antibodies in the panel.Then the effect on antigen binding is established empirically, toidentify those antibodies with can functionally pair to the variableregions of the other antibodies in the panel (FIG. 2). This methodidentifies pairing-compatible variable regions that are not immediatelyidentified by sequence comparison. Instead of using the partner variableregions derived of the antibodies in the panel, also other partnervariable regions can be used. For example, the heavy chain variableregion of each of the antibodies in the panel is combined with a set ofchosen light chain variable regions, for example, consisting of mainlygerm line encoded segments representative of one or more of the lightchain kappa or lambda gene families. Pairing-compatible variable regionsare then identified by screening the combinations for antigen bindingand scoring whether one common variable region provides antigen bindingfor the desired set of antibodies in the panel. These methods can bebased on assessment of antigen binding of individual combination of thevariable region genes, thus co-expression of two variable regions in thedesired antibody format, or of antigen binding of multiple combinationsof variable regions derived from co-expression in the same host cell.For example, two antibody heavy chain variable regions can be expressedinside the same host cell as Fd chain, and co-expressed with one lightchain, and antigen binding for both antibody binding sites assessed.Further, by differentially tagging the two heavy chains, for example,with epitope tags such as tags derived from c-myc, VSV, HA, etc., thepairing of the two H-L combinations can be followed. Such an approach issuitable for finding pairing-compatible variable regions if a limitednumber of starting antibodies is available and allows the screening oflarge collections of partner variable regions.

Examples of pairing-compatible variable regions are V-regions based onhighly homologous germ line segments, or V-regions that differ bychanges in the amino acid sequence (e.g., with somatic or othermutations, minor deletions, additions, substitutions). In such case theeffect of the exchange of the homologous region in the first antibodymay differ from the effect seen with the exchange of the homologousregion in the second antibody; e.g., there are cases where the affinityis changed to an allowable level for only one of the two antibodies, andcases where this occurs for both antibodies. In one embodiment, thepairing-compatible variable region comprises the light chain variableregion or part of the light chain variable region. In anotherembodiment, the pairing-compatible variable regions comprise the heavychain variable region or part of the heavy chain variable region.

Another embodiment of an approach to identify pairing-compatiblevariable regions in a panel of antibodies is the following. First thevariable region of each of the antibodies is co-expressed with a partnervariable region derived from the other antibodies in the panel, and ascreen carried out that will detect the presence of intact antibody(thus, not antigen binding). The formation of intact antibody indicatespairing between the two variable regions; if no intact antibody isretrieved, this will indicate that the two variable regions are notpairing inside the host cell. The screening can be used to identifyantibodies that display variable regions that cannot pair with oneanother in the chosen antibody format, i.e., as Fab fragments expressedin E. coli or as IgG molecules expressed in eukaryotic cells. Whenco-expressing the four variable region genes, only the cognateinteractions occur, and the variable region genes arepairing-compatible.

d. Antibodies with Pairing-Compatible Variable Regions From AntibodyLibraries

In certain embodiments, antibodies with pairing-compatible variableregions are selected from synthetic antibody libraries with a highprobability of identifying antibodies with such elements. Syntheticantibody libraries are collections of antibodies which have beensynthetically diversified (e.g., using site-directed mutagenesis orPCR-based gene synthesis using mutagenized oligonucleotides) in definedregions/locations within their variable regions. In one embodiment, thedesign of the diversity introduced into the primary antibody repertoireis such that at least a portion of a variable region and preferably acomplete variable region is not diversified, while the remaining areacontains the diversity (examples in FIGS. 3, 4(a), 4(c) and 4(d)).Examples of such libraries are libraries based on human variable regiongenes, for example, a set of 49 different heavy chain genes withdiversity introduced in the VH-CDR3, all combined with a single lightchain (H. R. Hoogenboom, et al. (1992) J. Mol. Biol. 227:381-388).Antibodies selected from such repertoires will contain by designpairing-compatible variable regions. Such repertoires can be created byrecombinant DNA methods and displayed on the surface of phage, cells,spores, ribosomes, or can be created in transgenic mice carrying onlypartial diversity in the V-gene composition. Synthetic diversity can beintroduced in all CDR residues, in a subset of CDR residues, i.e., thosewith significant solvent exposure, and can be designed to encode all ora subset of amino acids, i.e., those that are commonly observed innatural antibody CDRs. An example of such tailored antibody library,with a single heavy chain variable domain scaffold and a fixed lightchain variable domain, and with a limited number of heavy chain CDRresidues variegated with a limited number of encoding amino acids isdescribed in J. Mol. Biol. 338:299-310 and in WO 03/102157A2.Alternatively, to libraries with synthetic diversity in one variableregion, also libraries with natural diversity, or combinations ofnatural and synthetic diversity (e.g., synthetic diversity in CDR1 andCDR2 and natural diversity in CDR3) in one variable region may be used.

In one embodiment, antibodies with pairing-compatible variable regionsare obtained by first selecting an antigen-specific single-domainantibody, and then providing this with a second domain that will pairwith the first one to form a two-domain molecule (examples in FIGS. 4(b) and 4(e)). Single-domain antibodies are preferably isolated from invitro display repertoires made from single-domain repertoire of certainhuman variable region fragments, such as human VH or human VLrepertoires. In another embodiment, single domain antibodies areisolated from non-immunized, immunized or synthetic VHH repertoires,based on antibody heavy chain domains naturally devoid of light chains(e.g., camel, lama or some shark antibodies). Single-domain VH-basedantibodies with antigen-binding activity can be combined via recombinantDNA technology with a single, a small repertoire, a chosen collection ora large repertoire of light chains, preferably of human nature.Antigen-binding variants of single-domains now forced to contain apaired light chain, may be isolated using display technology based orequivalent methods. In another embodiment, single-domain VL-basedantibodies with antigen-binding activity are combined via recombinantDNA technology with a single, a small repertoire, a chosen collection ora large repertoire of heavy chains, preferably of human nature.Antigen-binding variants of single-domains now forced to contain apaired heavy chain, may be isolated using display technology-based orequivalent methods. In the embodiments of FIG. 5, the variants derivedfrom the same route of isolation will always share a variable regionsequence, thus will be able to provide functional pairing when broughtinto the context of pairing multiple variable regions.

If at least a portion of a variable region and preferably a completevariable region is not diversified, while the rest of the variableregion(s) contain the diversity, the selected antigen-binding antibodiescoming from such repertoires will contain by design pairing-compatiblevariable regions. In many of the approaches in the literature used forbuilding high affinity antibodies from synthetic antibody libraries,diversity in the initial library is built up throughout the antibodyvariable region genes and, in particular, in most of the six CDRs.Depending on the genetic make-up of these libraries, there will be ahigher or lower probability of identifying antibodies withpairing-compatible variable regions. Libraries can be designed to fitspecifically this new application, by introducing diversity in onevariable region only, and not further diversifying the shared variableregion, even in further affinity maturation processes. Preferably,libraries are used in which the diversity is restricted to the threeCDRs in one chain. The partner-variable region is then preferably one ora small set of germ line gene-encoded regions without any furtherdiversity. In the primary library or follow-up libraries, diversity canbe introduced in those areas of the antibody V-regions that are lesslikely to interact with the partner chain, so as to increase the chancesof finding antigen-binding antibodies with high affinity, yet wellpairing variable regions.

Antibodies with a high likelihood of containing pairing-compatiblevariable regions can also be enriched from antibody repertoires notbiased in their genetic make-up, by combinations of selections andre-shuffling of preferably the complete V-region of a given populationor clone (exemplified in FIG. 5). This will enrich for thoseantigen-specific antibodies with a high likelihood of containingpairing-compatible variable regions, for example, because they aretolerant in their pairing with the shuffled region yet retainantigen-binding, or because the shuffled region is less likely tocontribute to antigen binding. For example, an antibody Fab library isfirst enriched on antigen, and the selected heavy chains obtained afterone or more rounds of selection are then recombined with the selected orunselected light chain repertoire (dashed lines in FIG. 5), and selectedagain on antigen (FIG. 5, step 4). In this way the selected antibodyvariable heavy chain domains will have the propensity to bind to theantigen relatively independently of the light chain to which it ispaired. Antibodies to a first and second antigen can be identified byusing the above-described selection and re-shuffling experiment,followed by a screening as before, to detect antigen binding of theselected heavy chains in combination with a collection of light chains.One may then identify those antibodies that bind either the first or thesecond antigen relatively independently of the light chain, or in thepresent of a related light chain family member. Due to the dominance ofthe heavy chain in antigen binding in these antibodies, many of thelight chains are likely to functionally pair with the multiple heavychain variable regions. Co-expression of antibodies with apairing-tolerant variable region that is mediating antigen binding (suchas the VH), and in which the partner domain is not involved or notimportant for antigen binding (such as the VL), will similarly lead tothe formation of mainly or only functional binding sites.

In another embodiment, the invention describes a method to obtainantibodies with heavy and light variable regions that preferentially orin the best case, exclusively, pair to one another and not to therespective light and heavy variable regions of one or more otherantibodies, for example, those that are co-expressed in the same host.Such selection can be done by display methodology, but also using anintracellular selection route that relies on co-expression of antibodylight and Fd chains in the same cell, allowing competition between thechains, and rescue of the intended combination via phage display or anyother suitable route. The preferential or ideally exclusive pairing thatis encountered in faithful antibodies will aid in the formation ofmainly or only functional binding sites when such antibodies areco-expressed. This method essentially allows a high level of functionalantibody binding sites to form even when variable region genes are usedthat have very distinct compositions. A method for identifyingantibodies with desired pairing behavior based on competition selectionis described here. Antibodies are selected from a library of antibodyfragments, by carrying out a selection directly in a host cell thatco-expresses different antibodies. For example, when applied to usingbacteriophage libraries, this concept is the following: bacteria areprovided with a phage or phagemid genome that carries the genes encodinga Fab fragment in such manner that upon expression, one of the chainswill be anchored to a phage particle. In the same host cell, otherantibody light and/or heavy chain Fd fragments are co-expressed, forexample, the Fab genes encoding a given antibody, or any set of multipleantibodies. For example, consider co-expression of two Fabs in the samecell, one of which is anchored via its heavy chain (Fd fragment,essentially VH-CH1) to the phage coat protein. As a consequence of thisco-expression, competition occurs inside the same cell (in this case inthe periplasm) between the two light chains for the pairing to thephage-anchored Fd chain. Further, the soluble heavy chain of thecompeting Fab will be able to pair with both light chains present in thesame cell. In this system, phage particles with antigen binding activitywill occur with different types of pairings. First, if the correct lightchain will pair with its partner heavy chain on the phage only(exclusive pairing), and secondly, if the heavy chain on the phagesurface is dominant in antigen binding and tolerant for pairing,yielding antigen binding virtually irrespective of which light chain itpairs with. Functionally such antibody pairs will behave in the samemanner. In the case of the first situation, the lesser interactionsbetween the partners of the two respective antibody pairs, the higherthe proportion of functional Fab on phage. The method described can befurther biased towards antibodies with preferably an exclusive pairing,by providing tags on the chains and enriching or depleting forparticular combinations (e.g., depleting for those phage that carry thecompetitor light chains via a unique tag present on these chains). Thismethod when applied to the isolation of antibodies via the selection ofa phage library of Fabs, will yield a high frequency of antibodies thatwill have an appropriate pairing behavior and high functional yield whenproduced as mixture by co-expression. The use of competition-selectionto bias selected antibodies towards being co-expression compatible, mayalso be applied to other display libraries (e.g., yeast displaylibraries), and to in vitro library systems based on ribosome display ormRNA display (Puromycin system), with methods of screening or selectionof antibodies that recognize antigen as extensively described in theart. Further, the described method of competition-selection of antibodyfragments for improved pairing (or antigen-selection and compatiblepairing) using phage display can be readily translated into anintracellular (periplasmic) selection system based on protein-or enzymecomplementation. In such approaches, fragmented, complementary orself-inhibitory enzymes are used to drive the selection of interactingmolecules that are fused to the components of the selection system. Onlywhen there is an interaction of a minimal strength will the protein orenzyme become activated, and under appropriate selection conditions,will the cells survive. Such methods have, for example, been describedfor the enzymes beta-lactamase and DHFR, with its applications in theselection of antibodies or expressed cDNA fragments that display aparticular binding behavior. For example, competitive selection has beendescribed for the affinity maturation of antibodies in the TACZYMEsystem from Kalobios Inc. In the current invention, it is not theantigen binding but the pairing strength that can be made the selectiveforce for a given population of antibodies presented in such system.

In a preferred embodiment, the method is used to identify new antibodiesfrom phage libraries that show pairing-compatible variable regions withan existing antibody that has given variable region sequences. Theantibody with the known antigen specificity is cloned for co-expressionas Fab fragment in host cell that collectively express a phage displaylibrary of human Fab antibodies. This can be done by providing the Fabexpression cassette onto a plasmid that is compatible with the presenceof a phage or phagemid genome, such as the pBR322-based plasmid. Hostcells harboring this plasmid are then infected with the phage particlesencoding a library of human Fabs cloned into, for example, a phagemidvector such as pUC119, or a phage vector such as fd-tet-DOG1. While thecompeting Fab fragment is expressed, new phage particles are harvested(after helper phage infection if appropriate) from this culture. Theseparticles are used for selection on antigen, and the resulting phagereinfected into cells harboring the competitor Fab fragment. After a fewiterative rounds, the phage Fabs are screened for antigen binding in abinding assay; the pairing behavior between the reactive Fabs and thevariable regions of the competing Fab can be further tested byco-expression and binding assays. The preferred format for thisselection is the Fab format and not the scFv format, mainly because formost applications whole IgG-type antibodies will need to be establishedthat have interactions between the chains that harbor the variableregions that mimic those seen in the Fab format. FIG. 6 depicts anexample of how this method works for two Fabs competing with antibodiesin a phage library.

This method requires some optimization steps, for example, the use of aCH1-mutant with reduced affinity for its CL, and Fabs that do notdisplay an intermolecular disulphide bridge such that the pairing willremain noncovalent. Residues positioned at the CH1-CL interface regionmay be mutated such that affinity between these two domains is reduced,for example, 10-fold or 100-fold, and as a result in the Fab format thepairing of the variable domains will become more dominant in driving thetwo chains together. Antibodies selected from such mutated Fablibraries, or from Fv libraries in which there is no covalentassociation between the two variable regions, may be biased towardshaving a preferential pairing behavior.

In a further embodiment, the invention comprises the creation ofantibody libraries in which provisions are made to mediate uniquepairing between the heavy and light chains, such that they are unlikelyto interact with antibodies derived from a “regular” or non-purposelybiased composition. An example of such provision is a knobs-into-holesengineered CH3—CH3 pair, in which one domain is provided with an aminoacid with a large, bulky side chain (e.g., a tyrosine; the knob) thatpokes out into the interface region, while the other domain at theequivalent structural position, carries one or more mutations (e.g.,three) to create a hole into which the “knob” will fit. Examples of suchengineered domain interfaces have also been published for variableregions (Zhu et al. (1997) Protein Science 6:781-788). It was shown thatthe effects of domain interface mutants are context (antibody)dependent, which provides also an opportunity to engineer the variableregion domain interactions in an antibody-specific manner, in such waythat when multiple antibody variable gene pairs are allowed to pair,mainly or only the cognate pairings are retrieved. Alternatively,installing a disulphide bond between the domains may mediate apreferential pairing. Alternatively, charge replacements are introducedin the framework regions, or combinations of these with stericallycomplementary mutations, to disfavor mispairing with one, and/or morefavorable pairing with the other partner variable region. Selectionsystems for such mutant libraries have been described earlier, andinclude the selection of the domain libraries on antigen via phagedisplay of the paired variable regions (in scFv or Fab or, IgG format),or ribosome display of the scFv fragments, or selections based on theinteraction itself instead of that with antigen. An example of thelatter is described for selecting heterodimers of the immunoglobulingamma-1 CH3 domain (Atwell et al. (1997) J. Mol. Biol. 270:26-35), whichis applicable for the present invention as follows: on of the twovariable regions that should or should not interact (depending on whatone would like to select for, repulsion or attraction/pairing) isdisplayed on phage (preferably as VLCL or as VHCH1 chain), while theother is genetically tagged and produced in solution (preferably asVHCH1 or as VLCL). The interaction between the two variable regions canthan be selected for, using standard phage selection protocols andanti-tag reagents. Co-expression with a pair of non-tagged competitorvariable regions as described earlier can be used to drive the selectiontowards variable region pairs that exclusively pair with one another.

In another embodiment of selecting binding sites with appropriatepairing behavior, we describe here the use of antibodies derived fromVH-VH libraries on the one hand and VL-VL libraries on the other; or theuse of chimeric libraries in which elements (one or more CDR regions)are swapped between VH and VL. In another embodiment, the inventioncomprises the creation of two antibody libraries with such provisionsmade to mediate unique pairing between the heavy and light chains, suchthat when antibodies from these libraries are co-expressed, they willlikely preferentially pair with the right partner.

Cited libraries of antibodies can take various forms. As a source ofantibodies, a naive human library may be used, such as the antibodylibraries described by Griffiths (A. D. Griffiths, et al. (1993) EMBO J.12:725-734), Vaughan (T. J. Vaughan, et al. (1996) Nat. Biotechnol.14:309-314), or de Haard (H. J. de Haard, et al. (1999) J. Biol. Chem.274:18218-18230). Both heavy and light chains in these libraries arederived from the repertoires of rearranged V-genes derived from the mRNAof peripheral blood lymphocytes (PBLs) from unimmunized humans and are,therefore, highly diverse. Alternatively, as a source of antibodies animmunized host or patient with biased humoral response (e.g., patientswith infections, autoimmune diseases, etc.) is used. In immune librariesmade from a hapten-immunized animal, it was shown that many of theclones were promiscuous and allowed pairing of the originally selectedheavy and light chains with partner chains derived from other selectedclones. Thus, antibodies with pairing-compatible variable regions may bemore frequent in such immune libraries than in non-immune libraries.

Cited selection and screening technologies of recombinant antibodies andtheir fragments are well established in the field. Antigen-specificpolypeptides can be identified from display libraries by directscreening of the library, or can be first selected on antigen toincrease the percentage of antigen-reactive clones. The selectionprocess may be accomplished by a variety of techniques well known in theart, including by using the antigen bound to a surface (e.g, a plasticsurface, as in panning), or by using the antigen bound to a solid phaseparticle which can be isolated on the basis of the properties of thebeads (e.g., colored latex beads or magnetic particles), or by cellsorting, especially fluorescence-activated cell sorting (FACS). As willbe apparent to one of skill in the art, the antigen-specific affinityreagent may be bound directly or indirectly (e.g., via a secondaryantibody) to the dye, substrate, or particle. Selection procedures havebeen extensively described in the literature (see, e.g, Hoogenboom(1997) Trends Biotechnol. 15:62-70). Other publications describe theproduction of high affinity (nanomolar range) human antibodies from verylarge collections of antibodies, and the affinity maturation of theseantibodies by chain shuffling or other approaches (reviewed in, e.g., H.R. Hoogenboom, et al. (2000) Immunol. Today 21:371-378). Binding ofantibodies to their respective antigens may be carried out usingantibody-based assay techniques, such as ELISA techniques, Westernblotting, immunohistochemistry, Surface Plasmon Resonance (SPR)analysis, affinity chromatography and the like, according to methodsknown to those skilled in the art (see, for example, Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor Laboratory Press). These techniques are viable alternatives tothe traditional hybridoma techniques for isolation of “monoclonal”antibodies (especially when human antibodies are required), which areencompassed by the present invention.

The following describes possible embodiments of exemplary assays forbinding assays: ELISA. Polypeptides encoded by a display library canalso be screened for a binding property using an ELISA assay. Forexample, each polypeptide is contacted to a microtiter plate whosebottom surface has been coated with the target, e.g., a limiting amountof the target. The plate is washed with buffer to removenon-specifically bound polypeptides. Then the amount of the polypeptidebound to the plate is determined by probing the plate with an antibodythat can recognize the polypeptide, e.g., a tag or constant portion ofthe polypeptide. The antibody is linked to an enzyme such as alkalinephosphatase, which produces a colorimetric product when appropriatesubstrates are provided. The polypeptide can be purified from cells orassayed in a display library format, e.g., as a fusion to a filamentousbacteriophage coat. In another version of the ELISA assay, eachpolypeptide of a library is used to coat a different well of amicrotiter plate. The ELISA then proceeds using a constant targetmolecule to query each well.

Surface Plasmon Resonance (SPR). The binding interaction of a moleculeisolated from library of diversity strands with a target can be analyzedusing SPR. For example, after sequencing of a display library memberpresent in a sample, and optionally verified, e.g., by ELISA, thedisplayed polypeptide can be produced in quantity and assayed forbinding the target using SPR. SPR or Biomolecular Interaction Analysis(BIA) detects biospecific interactions in real time, without labelingany of the interactants. Changes in the mass at the binding surface(indicative of a binding event) of the BIA chip result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance). The changes in the refractivity generatea detectable signal, which are measured as an indication of real-timereactions between biological molecules. Methods for using SPR aredescribed, for example, in U.S. Pat. No. 5,641,640; Raether (1988)Surface Plasmons, Springer Verlag; Sjolander and Urbaniczky (1991) Anal.Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol.5:699-705 and on-line resources provide by BIAcore International AB(Uppsala, Sweden). Information from SPR can be used to provide anaccurate and quantitative measure of the equilibrium dissociationconstant (K_(d)), and kinetic parameters, including k_(on) and k_(off),for the binding of a biomolecule to a target. Such data can be used tocompare different biomolecules. For example, proteins encoded by nucleicacid selected from a library of diversity strands can be compared toidentify individuals that have high affinity for the target or that havea slow k_(off). This information can also be used to developstructure-activity relationships (SAR). For example, the kinetic andequilibrium binding parameters of matured versions of a parent proteincan be compared to the parameters of the parent protein. Variant aminoacids at given positions can be identified that correlate withparticular binding parameters, e.g., high affinity and slow k_(off).This information can be combined with structural modeling (e.g., usinghomology modeling, energy minimization, or structure determination bycrystallography or NMR). As a result, an understanding of the physicalinteraction between the protein and its target can be formulated andused to guide other design processes.

Homogeneous Binding Assays. The binding interaction of candidatepolypeptide with a target can be analyzed using a homogenous assay,i.e., after all components of the assay are added, additional fluidmanipulations are not required. For example, fluorescence resonanceenergy transfer (FRET) can be used as a homogenous assay (see, forexample, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos etal., U.S. Pat. No. 4,868,103). Another example of a homogenous assay isAlpha Screen (Packard Bioscience, Meriden Conn.). Alpha Screen uses twolabeled beads. One bead generates singlet oxygen when excited by alaser. The other bead generates a light signal when singlet oxygendiffuses from the first bead and collides with it. The signal is onlygenerated when the two beads are in proximity. One bead can be attachedto the display library member, the other to the target. Signals aremeasured to determine the extent of binding. The homogenous assays canbe performed while the candidate polypeptide is attached to the displaylibrary vehicle, e.g., a bacteriophage.

Automated screening. The methods and compositions provided herein arealso suitable for automated screening of diversity libraries for findingclones with likely pairing-compatible variable regions. For example, adisplay library of Fabs or scFvs can be screened for members that bindto a target molecule. The library can be screened directly or firstselected on antigen once or several times. Binders from a first round ofscreening can be amplified and rescreened, one or more times. Bindersfrom the second or subsequent rounds are individually isolated, e.g., ina multi-well plate. Each individual binder can then be assayed forbinding to the target molecule, e.g., using ELISA, a homogenous bindingassay, or a protein array. These assays of individual clones can beautomated using robotics. Sequences of the selected clones can bedetermined using robots and oligonucleotide primers that allow to readthe variable region sequences of the selected clones. Results of theassay and the sequences can be stored in a computer system and evaluatedby eye or by using software, e.g., to identify clones which meetparticular parameters (e.g., for binding affinity and/or specificity,and for sequence homology).

e. Forcing Appropriate Pairing of Antibody Variable Regions Via Mutationand Selection

There are instances where antibodies with given variable regionsequences, antigen specificity and affinity are available, but where nopairing behavior can be achieved with the existing sequences. Some ofthe methods mentioned earlier can be applied to solve this, inparticular, the screening of a combinatorial panel of variable regionpairs to find fortuitously compatible pairs, or the selection of newantibodies that do have the desirable pairing behavior, for example,using competition selection with one of the antibodies of definedspecificity. In those instances where this is not a desirable option andpreferably the existing antibodies are used, the following methods maybe used to create pairing-compatible variable regions for the set ofantibodies to be produced as an Oligoclonic™ mixture.

First of all the pairing can be biased by using single-chain Fv variantsof the antibodies. The provision of a linker between heavy and lightchain variable region will increase the chance that the two domains willpair with one another, instead of pairing with unlinked molecules orwith other single chain Fv molecules of the same or differentspecificity present in the same cell. If such molecules are fused to Fcregions and co-expressed in the same host cell, the result is a mixtureof scFv-Fc molecules which are paired via the heavy chain Fc region,forming monovalent and bispecific molecules. There is also analternative solution that does not rely on pairing in the scFv format.With a set of, for example, three given antibodies, an antibody mixtureconsisting essentially of IgG-formatted molecules can be made by makingthe variable region genes compatible with one another. First thesequence of the antibody light chains is determined, and the chain thatis the most common to the sequence of the two other light chain variableregions, or the closest to its germ line amino acid sequence identified.For the two antibodies that carry the different light chain, a libraryof heavy chains is created that is diverse in the CDRs including theCDR3 that produces a substantial fraction of the interactions betweenheavy and light variable region sequences. These heavy chains arecombined with the chosen, non-mutated light chain in a format thatprovides expression and screening, or display and selectioncapabilities. In such manner, the two remaining antibodies are forced toaccept the new light chain, which could affect pairing and affinity; theprovision of mutations in the heavy chains and the selection (eitherseparately as scFv or Fab fragments, or as Fab in competition with theiroriginal light chain in a method described above for competitionselection), will enrich for variants that have corrected a possibledeficiency in pairing efficiency and/or affinity loss.

f. Antibodies with Pairing-Compatible Variable Regions from TransgenicMice

It is possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production.Transfer of the human germ-line immunoglobulin gene array in mutant micethat carry a homozygous deletion of the antibody heavy chain joiningregion (JH) gene and, therefore, do not anymore produce murineantibodies, results in the production of human antibodies upon antigenchallenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. U.S.A.90:2551-255 (1993); Jakobovits et al., Nature 362:255-258 (1993).Antibodies with pairing-compatible variable regions may be identifiedfrom panels of antibodies made in these animals, or from such antibodiesand antibodies derived from other methods. It is envisaged thatantibodies with pairing-compatible variable regions may be identifiedeven more readily in transgenic mice carrying only the heavy or only thelight chain locus, and only a single or a limited set of chosen partnerchains; in that case immunization would lead to the generation ofantibodies which all carry a compatible common chain. Antibodies withpairing-compatible variable regions are then identified using themethods described herein. The efficiency with which such antibodies canbe identified can be further increased by reducing the extent of somatichypermutation of the partner chain or chains. This can, for example, bedone by removing regulatory sequences surrounding the variable regions,or by mutating the variable region codons such that the gene becomes aless likely substrate for the cellular hypermutation machinery, or byharvesting the B-cells earlier after immunizations.

One further approach is to combine the heavy chains of the threeantibodies with a repertoire of highly diverse light chains, and screenthe pairings, if necessary after selection on antigen, for light chainsthat maintain functional pairing (and antigen binding) and share acommon sequence. This can be readily carried out using automatedfacilities for high throughput ELISA screening and sequencing, aspresented earlier.

g. Uses of Antibodies with Pairing-Compatible Variable Regions

Antibodies with pairing-compatible variable regions have manyapplications. It is disclosed herein that the preparation of a desiredfunctional antibody mixture is feasible when the composition of thevariable heavy or light chains of the various antibodies is carefullyselected to contain antibody variable regions that carrypairing-compatible variable regions such that the pairing of theantibody variable regions yield predominantly functional binding sites.After selection of antibodies with pairing-compatible variable regionsas described above, the antibody variable region genes can be clonedinto expression vectors that will direct the expression of an antibodyof the desired format, e.g., IgG, IgA, IgM. In one embodiment, theinvention describes the production of mixtures of antibodies through theco-expression of variable region genes operably linked to constantregion genes, in which these variable region genes encode differentantibodies with pairing-compatible variable regions. Without theselection of appropriately pairing antibodies with pairing-compatiblevariable region, co-expression would lead to the formation of a mixtureof antibodies with many non-functional heavy-light chain combinations.When appropriate pairing-compatible variable regions have been defined,a high level of functional antibody combining sites will arise. In oneembodiment, the heavy chain variable region is operably linked to thefirst domain of the heavy chain constant region, followed by a hingeregion, followed by the remaining domains of the heavy chain constantregion. The variable region of the light chain on the other hand isoperably linked to an appropriate constant domain of the kappa or lambdafamily.

In a preferable embodiment, the pairing-compatible variable region is anidentical light chain. In that case the co-expression of this lightchain and, for example, two different heavy chains derived fromantibodies with as pairing-compatible variable region the full lightchain, in the same cell will yield a mixture of the two expectedbivalent molecules and one bispecific molecule. Similarly, whenco-expressing this light chain with more than two heavy chains derivedfrom antibodies that all have functional antigen binding sites whenpaired to that same light chain, the mixture will contain in a certainfraction each of the bivalent molecules, and a number of bispecificmolecules with combinations of all binding sites, e.g., three when threeantibody heavy chains are introduced, six when four antibody heavychains are introduced, ten when five antibody heavy chains areintroduced, etc. In this case, the affinity of the monomeric bindingsites in these various species is expected to be very similar to theaffinity of the original binding sites. In another embodiment,antibodies share a pairing-compatible variable region, but the sequenceof this element is different between the two antibodies and, uponswapping, the affinity of one or both of the antibodies may be altered.If such antibodies are used for co-expression, the final antibodymixture will contain antibodies with the original and the alteredbinding affinity in all of the species that were mentioned above. In thepreferred embodiment, such antibodies share a compatible common lightchain. In another embodiment, antibodies share a compatible common heavychain. The expression levels of the individual components can be chosenor can be manipulated to alter the fraction of the species of antibodiescontaining that component.

2. Protein Mixtures with Optimally Paired Variable Regions

Using the methods according to the invention, antibodies with a pairingbehavior suitable for the preparation of well-defined biopharmaceuticalmixtures are obtained. Traditionally before use for human therapy,protein drugs are expressed and purified to homogeneity, consisting ofone major molecular species. In some cases, therapy is more efficaciouswith combinations of proteins or other drugs. This invention describesmethods to make a proteinaceous mixture that will contain at least twomajor molecular species, composed of at least three variable regions,and such that some variable regions pair to form a functional bindingsite. The large-scale manufacturing of the proteinaceous mixture is aprerequisite for their clinical use, and a simple purification procedureis an important feature of the development process. The presence ofinappropriately paired variable regions would inevitably lead to a morecomplicated purification procedure. In one embodiment, the genesencoding the components of the two proteinaceous compounds areco-expressed in the same host cell, and the different major molecularspecies that are present in the mixture and have a functional bindingspecificity purified using biochemical/biophysical techniques well knownin the art. In one embodiment, the method is used to make a mixture of adefined number of antibodies. The major molecular species that compriseone or more different binding specificities could share a minimalproportion of their encoding genetic information (e.g., an Fc region, acommon tag, or another shared domain or feature); such shared featurewill provide a common mechanism/assay for following the individualcompounds in the mixture. In another embodiment, the major molecularspecies are preferentially co-purified due to a similarbiophysical/biochemical behavior, or due to a shared domain thatmediates co-purification (e.g., an Fc). In another approach, the majormolecular species are fused to a subunit of a protein such that they canmultimerize with each other (e.g., CH2-0H3 region). The invention alsoprovides biopharmaceutical mixtures produced using this method. Thepreferred application is the co-expression of antibodies, with thechoice of the V-genes and pairing behavior between VH and VL domainssuch that mainly or only functional binding sites are made, and thepurification of the mix can occur via the shared feature, an Fc region.Methods for purification of immunoglobulin are well known in the art,including protein A, protein G and other affinity matrices. Otherproteinaceous mixtures that could be envisaged to have paired variableregions are fusion proteins between antibodies or antibody fragments andother molecules, single domain antibodies derived from camel, llama orengineered single domain antibodies from murine or human variable regiongenes, receptor extracellular domains, peptides, proteins equipped withan engineered binding site, or cytokines. Preferably, the proteinaceouscompounds share a feature (like by further fusion to an immunoglobulinFc region; methods well known in the art), such that they can beco-purified using the same procedures. The optimal pairing of thevariable regions in the different proteinaceous compounds will also leadto an optimal level of functional binding sites on these compounds, thusminimizing the number of purification steps required to obtain theactive component of the protein mixture.

3. Selecting Antigen-Specific Proteinaceous Compounds Using Mixtures ofEncoding DNA

In a preferred embodiment, the proteinaceous compounds are antibodies.In the invention antibodies are identified in collections or pools ofgenetically diverse antibodies, in which the pairing of the variablegenes is optimized in such manner that upon co-expression of at leasttwo antibodies inside the same cell an optimal pairing arises, providinga maximal amount of functional binding sites. In a preferred embodiment,the pairing of all binding sites is optimized due to the use of a sharedvariable region gene, preferably the light chain. The diversity of theother elements in the library will be such that antibodies with highaffinity can still be selected. Due to this choice of the genetic makeup of the variable regions, the pairing of the antibody variable regionswill be such that a very high level of functional binding sites will bepresent when multiple variable regions forming more then one antibodybinding site are contacted with one another, for example, when expressedin the same cell. In one embodiment, first a library or collection ofdifferent antibody heavy chain genes is made, and cloned into aneukaryotic cell expression vector. This library is introduced into hostcells in such a manner that each host cell will be making multipledifferent antibody heavy chains. In a preferred embodiment,“anti-repressor elements” (Kwaks et al., 2003, Nat. Biotechnol. 21:553)are cloned at one or both ends of the antibody heavy chain gene. Suchelements confer stable and high level expression of a given transgene asshown in this citation, and in this invention we describe its use tomediate stable and high level expression for each individual copy of thetransgene (see also below).

In one embodiment of this invention, depicted in FIGS. 7-8, the variableregion or regions with optimized pairing behavior for the other variableregions is or are also genetically encoded in an appropriate expressionvector, and introduced into the host cell, either before, during orafter the introduction of the other variable region. The expressioncassette with the variable regions can also be part of a viral systemsuch that high levels of transfection/infection efficiency can beachieved. In the case that the pool of first variable regions areantibody heavy chains, the second variable region with optimized pairingbehavior can be one or more light chains. The host cells which aretransfected with both partners of the pairing, e.g., the mix of antibodyheavy chains and set of light chains, are expanded and grown underconditions which allow the expression of heavy chains and light chains.Preferably only one light chain is used, as exemplified in FIG. 7. Forexample, the expansion can occur in tissue culture wells, in such amanner that the tissue culture wells will contain between 10-1000different originally transfected clones, each of the clones expressingmultiple pairings of the antibody variable regions. Antigen-specificantibodies can be retrieved amongst these clones and wells by variousmethods, preferable by ELISA or equivalent test of the antibody mixturesof each well (see also earlier description of binding assays). If stabletransfection is used, with the possibility to select transfected celllines for stably integrated copies of the antibody encoding DNAs, therelevant antibody or antibodies may be cloned via limiting dilution.Alternatively, the DNA encoding the relevant antibody variable genes canbe retrieved by amplifying and sequencing the antibody genes from thecells in the well using methods know in the art. If required, theantibody-heavy chain encoding DNA can be also amplified, recloned forexpression in the same system, the DNA amplified and then used to repeatthe transfection, expression and screening experiment. With this cycleof transfection and screening, after a few rounds, an antigen-reactiveantibodies start dominating the population. At every round, thecomplexity of the mixture produced by an individual cell can be reducedby reducing the complexity of the DNA introduced into the cell, toeventually become a oligoclonal population. From the transfected wells,the antibody's V-gene can be rescued directly (e.g., via PCR) andfurther analysis and/or screening in this system, eventually atconditions that provide expression of the monoclonal antibody.Alternatively, the variable regions from reactive wells can be clonedinto other systems for rapid screening of the binding specificity of theindividual pairs of variable regions, e.g., via bacterial expression ofantibody fragments or whole IgG, expression in other hosts, via in vitrodisplay methods, bacteriophage display methods etc.

In another preferred embodiment, the heavy chains are secreted by thehost cell into the supernatant, where they can be reconstituted intofunctional antigen binding fragments, by the addition of and pairingwith a partner light chain. This can be a small family of relatedchains, but preferably one chain only. In this approach, cells are usedthat do not prevent secretion of the non-paired heavy chain. Thisembodiment is depicted in FIG. 8. Drosophila S2 cells have beendescribed that contain a BiP (Binding Protein) homologue, hsc72, thatspecifically interacts with immunoglobulin heavy chains, but does notprevent their secretion. Alternatively, the heavy chains will need tocarry amino acid mutations in such a manner that cells that normallyretain heavy chains when they are not paired to light chains, will notmediate retention anymore. For example, mutations can be provided foror, selected within, the major recognition sites for BiP sites which arelocated in the heavy chain CH1 domain. For example, the CH1 domain canbe replaced (e.g., by a CL or CH3 region) as long as the light chain canpair with this form of the molecule (or other variants, see also sectionon antibody cross-over variants), or mutated to avoid retention by BiP.The results of such variations are that the different heavy chains aresecreted by the host cell. The chains are then reconstituted with one ormore partner chains carrying the partner variable region(s). Methods toestablish this have been extensively reviewed in literature on thebiochemical analysis and assembly of antibody molecules.Antigen-reactive variable region pairs can be identified in the same wayas described for the other embodiment.

In yet another embodiment, antibody the first partner of the two pairedvariable regions (such as the heavy chain for an antibody) is anchoredonto a eukaryotic cell surface, and the other variable region providedby expression in the same host cell or via reconstitution on thecell-surface. This set-up allows a direct screening for antigen-bindingon the host cell surface, for example, via flow cytometry withfluorescently-labeled antigen, or a direct selection, for example, viacell sorting methods.

Methods to identify antigen-reactive antibodies from B-cell populationshave been described in the literature and can be applied to thesetransfection-based systems also. In such described systems, randomcombinatorial diversity is sampled, and antibody variable gene pairingis also not optimized or biased. Use of such random combinatorial pairsof variable regions does not guarantee that upon production of anantibody mixture, the pairing will be optimal; on the contrary,mispaired variable regions will be a substantial fraction of theproduced proteinaceous compounds. In this invention an important elementis that this random combinatorial diversity is limited, by reducing thediversity of one of the variable region genes. The diversity that ispresent in the resulting paired repertoire originates mainly from one ofthe variable regions. Preferably, it is one or a small set of lightchains. As a consequence, in the iterative process of selecting theantigen-reactive variable regions, only one of the two partners of thepair will need to be identified. It is not necessary to retrieve boththe heavy and light chain variable region sequence from the same cell.Another important difference is that multiple antibody genes areintroduced and expressed from the same host cell. When using randomdiversity, such a feature would lead to a multiplication of thediversity and reduction of the quantity of the individual combinationsto the extent that detection let alone cloning of the responsibleantibody gene combination would become very difficult, if notimpossible. Consider the case in which the cell would be making multiplecombinations of heavy and light chain pairs, then the chance to retrievethe correct combination of the antibody that mediates antigenreactivity, would be become smaller as the cell is making a highernumber of different chains. If the cell would be expressing tendifferent heavy and light chains, the combinatorial diversity generatedby this one cell would be a 100 different types of antibody bindingsites; only 1/10 of the antibody variable genes amplified from such cellwill be the relevant one, thus the chance to be able to clone thecorrect antibody genes is very low. As a consequence of this reducedcombinatorial diversity in the present system, there will also be ahigher quantity of each of the individual antibodies, which makes a moresensitive detection possible. Thus, in this invention the expression ofthe different antibodies in the same host cell is a desired feature.First as explained above, it is an important feature for theantigen-selection system to find antigen-reactive antibodies when usingtransfected cell populations. Secondly, the invention is directedtowards the production of mixtures of proteins and more in particular,antibodies or their fragments, which requires optimal pairing of thevariable regions, in particular, when producing such mixtures byco-expression in the same host cell. In the preferred method describedabove, co-transfection of variable region genes inside the same cellsleads to the expression of multiple antibodies in the same host cell.The methods are thus useful to select individual antibody variableregion pairs that are reactive with a given target epitope, but also toselect a mixture of different variable region pairs all reactive with agiven target epitope (in the process of the screening, multiple antibodyvariable region pairs will be selected or identified, but when iteratingthe process, these antibodies are likely to be eventually mixed and endup in the same host cell). Further if the screening or selection of themixture is carried out with targets with multiple epitopes, or multipletargets, the mixture can also contain antibodies to multiple epitopes ortargets, yet with co-expression-compatible pairing of the variableregion genes.

The invention is also suitable for the screening of mixtures of proteinswith paired variable regions that have a defined binding specificity(FIG. 9). The genes encoding these compounds are introduced as a mixtureinto a host cell as above (in FIG. 9 examples is given of ten differentantibodies), and individual clones that have integrated some or multiplecopies of the genes encoding the various variable regions expanded. Inthe way described above, applied to antibodies, the supernatants of theresulting cell lines are screened for reactivity towards the variousantigens. The levels of each of the individual antibody pairs may vary,and, when the antibody format is the IgG isotype, also the level of thebispecific antibodies resulting from the co-expression may be highlyvariable. Cells that secrete the mixture comprising the desiredcomposition are identified and used as a stable production host for thismixture. The invention provides a method to quickly screen hundreds ofmixtures of different antibodies. The optimized pairing of the heavy andlight variable regions will secure a high level of functional bindingsites in the antibodies present in such mixtures.

4. Antibody-Based Compounds with Paired Variable Regions and Cross-Overor Mutations in the Constant Regions

The pairing of the variable and constant regions of an antibody can befurther engineered as follows, by crossing-over domains. Antibodies aremade by crossing-over or swapping or replacing elements within the Fabregion of the antibody (or the antibody heavy chain Fd region and theantibody light chain region), and combining the appropriate elements toestablish a binding site in the context of an immunoglobulin molecule(examples are given in FIG. 10). In its simplest format, the L chain andH chain Fd region are swapped. A VL-CL-hinge-CH2-CH3 chain is thuspaired to a VH-CH1 domain. In a second format, the constant region genesbetween H and L are swapped. In another form, the CH1 is replaced by aCL. In another form, the VH and VLs are swapped. In another form, one ormore of the CDR regions between VH and VL are swapped. The pairingefficiency can be monitored in such cross-over variants, such thatsuitable combinations of non-cross-over antibodies with cross-overantibodies, or combinations of different cross-over antibodies, can beused to mediate optimal pairing when making mixtures of at least twoantibody molecules (with antibody also including here cross-overvariants as described above). In another form the effect of mispairingbetween different VHs and/or VLs is reduced by linking the VH and VL viaa linker to a single-chain Fv variant, which will favor the associationbetween these two domains. Alternatively, the pairing between variableregions can be manipulated by the introduction at the appropriatepositions of cysteines which upon pairing of the variable heavy andlight variable domains can form a disulphide bridge. The invention alsoprovides methods for selecting antibody fragments that will bind antigenin an appropriate cross-over format, by selecting from appropriatelyformatted libraries, or by screening one or more antigen-bindingantibodies for the activity in the cross-over format. Antibodies inwhich the CH1 domain is not part of the heavy chain may be secreted asfree molecules not paired to light chains, allowing alternativeapproaches for the production of antibodies and new fusion formats.Antibodies in which the variable regions are swapped may be functionallynon-equivalent and yield a more diverse, unnatural or different spectrumof antigen-binding or biological activity (the positioning of the heavyand light chain variable regions is expected to not always be completelyequivalent). Besides effects of the exchange of the heavy and lightchain genes on affinity and/or specificity, the swapping may alter theantibody flexibility and impact the biological behavior. Finally, anantibody binding site with chimeric VH-VL regions (with CDR or FRregions swapped between the two variable domains) may also yield analternative, possibly larger but structurally non-overlapping set ofantibody paratopes.

Secondly, selective engineering of the constant regions or theinteraction of variable regions with constant regions may also affectthe pairing behavior of the variable region genes. By modifying theantibody heavy chain constant region, the fraction of functionalbispecific antibodies can be increased or decreased. In this approach,antibody heavy chains can be engineered to drive hetero- orhomodimerization. This can be done by introducing stericallycomplementary mutations in the CH3 domain interface, for example, as hasbeen described in the literature for increasing the percentage offunctional bispecific antibodies in the mixture of antibodies arisingfrom the co-expression of two heavy and two light chains. The pairing ofthe antibody binding site variable region may thus be influenced by thepairing of variegated constant regions, of heavy and light constantregion domains.

5. Extracellular Pairing of Proteinaceous Mixtures

This invention provides a method for making whole antibodies using an invitro pairing procedure of heavy and light chains produced in differenthost cells. In one embodiment, one of the two antibody chains isexpressed in a first host cell and the other chain is expressed in asecond host cell (FIG. 11A). The antibody chains are then broughttogether under conditions in which pairing of the two domains willoccur, thus outside of the cell. In one embodiment, the pairing occursin vitro, with purified chains and under conditions that are optimizedfor the pairing of the desired variable regions. In another embodiment,the expression occurs via the use of one or two dummy-chains,temporarily paired to the respective variable regions, removing thedummies from their partner via a mild and controllable process, andpairing the appropriate unpaired variable regions to one another to forma functional binding site. In one embodiment applied to antibodies, thisassociation is made easier by using heavy-light chain pairs mutated inone or the other chain to facilitate the process of the pairing, e.g.,mutated in the cysteine residue that normally forms the bridge between Hand L chains (either both mutated, for example, to Ser, or only onemutated and not the other), or mutations that have altered the affinityof one chain for the other or, preferably, mutations in the dummy chainused for the temporary pairing, in particular, the one that pairs withthe heavy chain; thus such dummy light chain will pair with a native,non-mutated heavy chain, and may carry mutations such that it can bereadily removed from the purified antibody.

An extension of this concept is that it is possible to produceantibodies using universal antibody chains (FIG. 11B). The inventionprovides a method for expressing a shared, invariant variable regioncontained into the appropriate chain format (e.g., a VL-CL light chain)in a given host cell, and the other chain (e.g., a heavy chainconsisting of VH-CH1 or VH-CH1-hinge-CH2—CH3) that is dominant in orprovides most or all of the specificity, in another host cell. Forproduction of two antibodies, three chains need to be made, which can beassembled in vitro to form two different antibodies. For example, if thelight chain is identical, only one VL-CL domain will have to be made,and two VH-containing heavy chains. These can then be assembledextracellularly, preferably in vitro. Pairing of the variable regionswill have to be optimal such that the proteinaceous mixture yields ahigh level of functional binding sites. The light chain can be useduniversally for all antibodies that will accommodate it (and antibodiesaccordingly selected if required). The heavy chain can be expressed inmammalian cells to provide a suitable glycosylation; for the lightchains any suitable expression host cell can be chosen. When using thisinvention with the cross-over variants described in the previoussection, in which the light chain is fused to the hinge and Fc, and theheavy chain variable region is provided as the lightest chain (as VH-CH1or VH-CL), an important advantage of this set-up is apparent: the lightchain fused to the Fc (depicted as “constant” chain in FIG. 11B), withits functionally important glycosylation features, can be made as theuniversal chain. The heavy chain can carry the dominant features for thespecificity, and a mixture of heavy chains which will mediate differentbinding specificities can now be made in a different host cell that doesnot need to provide glycosylation. Such feature makes the production ofmixtures possible in two steps: a cheaper prokaryotic expression can beused to make mixtures of variable regions each encoding a unique bindingspecificity, while the more expensive production of the other variableregion that also requires most fine analysis, can be done in aeukaryotic host. All antibodies that can pair with the latter variablegene without inflicting their overall specificity and affinity, can beproduced by extracellular pairing with the same universal chain. Thelatter can be designed to be optimized for pharmaceutical applications:a broadly expressed, relatively common variable region, with a minimalnumber of MHC Class II epitopes, of human origin, and germ line insequence. This procedure of mixing can be done with separate heavy chainmixtures or with a mix of the different heavy chains; when applied tothe IgG format as depicted in FIG. 11B, the result is an antibodymixture without or with bispecifics, respectively. Manual mixing andpairing of variable region genes further provides much more control overthe pairing, it can be done in a stepwise manner, per antibody, pergroup of antibodies etc. For some applications, for example, where thereis an absolute necessity to avoid the formation of bispecific antibodiesin a complex mixture with three or more antibodies, this method has anadvantage over the cell line-based approach.

6. Controlling the Expression of Variable Regions in the Context of theProduction of Multiple Pairing Variable Regions in the Same Host Cell

Nucleic acids encoding variable region, e.g., from antibodies, can beco-expressed in the same cell to make mixtures of different functionalbinding sites. With appropriate pairing behavior, a high level offunctional binding sites will be present. It will however also beimportant to control the expression of the individual variable regionsand their expression ratios, because this will effect the composition ofthe final antibody mixture. The expression level and the stability ofthe expression is a function of the site of integration of thetransgene: if the transgene is integrated close to or withininaccessible chromatin, it is likely that its expression will besilenced. In this invention, we describe the use for the production ofmixtures of antibodies in the same cell, of elements that, when flankingthe antibody genes, will increase the predictability of the expressionlevel, the yield, and improve stability. Such elements can, for example,do this by counteracting chromatin-associated gene repression. Suchanti-repressor elements provide a high level of predictability ofexpression, high levels of expression and stable expression overtime, ofthe antibody mixture (Kwaks et al., 2003, Nat. Biotechnol. 21:553). Suchelements confer stable and high level expression of a given transgene asshown in this citation, and in this invention we describe its use tomediate stable and high level expression for each individual copy of amixture of transgenes, encoding multiple variable regions. A variety ofsuch elements and other systems to achieve a similar result have beenidentified in the art, including Locus control regions (LCRs), chromatinopening elements, artificial chromosomes (e.g., ACE technology fromChromos Molecular Systems Ltd.), and Ubiquitous Chromatin OpeningElements. For example, LCRs are transcriptional regulatory elementswhich possess a dominant chromatin remodeling and transcriptionalactivating capability conferring full physiological levels of expressionon a gene linked in cis, when integrated into the host cell genome. Inthe following section, the invention is described for “anti-repressorelements” but other, different control elements such as the onesmentioned and inasmuch as they provide the opportunity to regulate thehigh-level expression of multiple genes, may be equally suitable toachieve a controlled expression of the different variable regions.

In one embodiment of the present invention, antibody mixtures are madefrom variable region pairs in which one dominates the binding, and theother is a shared variable region. In a preferred embodiment, the firstvariable region one is the heavy chain, and the second is the lightchain. In the preferred embodiment, at least one of the antibody heavychains is flanked by one anti-repressor element, or by two identical ortwo different anti-repressor elements located at either end of the heavychain gene; in another embodiment, more than one or possibly all of theheavy chain genes that need to be expressed are flanked byanti-repressor elements. In one embodiment, the heavy chains are basedon the same plasmid, in another they are on separate plasmids. Inanother embodiment, CHO cells are used as host; in another embodiment,PER.C6 cells are used.

The manufacture of mixtures of antibodies expressed in the same cellline will require appropriate variable region pairing and also a stableexpression level of all of the antibody chains involved, as well as astable ratio of the various chains, in such manner that the resultingantibody mixture after manufacture even at GMP conditions, has a stablecomposition. Such stable compositions can then translate into stablebiological activity and stable toxicity profile. If the expression ofonly one antibody chain would change, it could affect the compositionand, therefore, also alter its biological activity. The provision ofelements that yield a more predictable and copy-number associatedexpression level is also important to build cell lines that expresssimilar or even equimolar levels of different antibodies. If, forexample, five antibody heavy chains have to be expressed, it will bevery difficult to build a cell line that expresses all of these chainsat similar quantities when using a random integration and selectionapproach without the anti-repressor elements. By using such elements, ahigher copy number of antibody chains can be introduced withoutcompromising the stability of the resulting cell line. Thus, multipleantibody heavy chains can be introduced, where the number of integratedcopies for each heavy chain will also to some level reflect its absoluteexpression level. With such elements it will be much easier and morerapid to alter the ratios of expression levels between the heavy chains,for example, by manipulating the ratios of the DNAs encoding the heavychains at the time of the transfection.

This also explains the preferred incorporation of such anti-repressorelements in vectors to be used for creating antibody libraries andselect antigen reactive antibodies from these pools (see section 4);anti-repressor elements preferably inserted in the expression vectorsthat incorporate the heavy chain, on FIGS. 7, 8 and 9.

7. Expression Systems for Multiple Variable Regions in the Context ofthe Production of Multiple Regions in the Same Host Cell

When expressing multiple variable regions inside the same cell, maximalproductivity will be achieved only if the partners that need to bepaired are co-expressed at an equivalent level, such that there islittle chance on what is essentially waste: the non-paired variableregion. The composition of the mixture is influenced by manipulating anyone of the parameters that affect the expression level achieved in thehost cell. The expression level of a given component is a function ofmany factors including the regulatory sequences that drive theexpression of the component, when the component is a heavy chain alsothe expression levels of the light chains, the choice of the host cell,the method of expression (transient or stable), and, for stableexpression, the copy number and site of integration. The expressionlevels can further be affected by many parameters including choice ofthe transcriptional regulatory elements (including choice of promoter,enhancer, insulators, anti-repressors, etc.). The expression of the twolight and heavy chains of the antibodies that are to be assembled fromthe mixture of the chains can be done independently for each of thechains, or made dependent from each other.

The expression vector or vectors comprising the antibody genes ofinterest contain regulatory sequences, including, for example, apromoter, operably linked to the nucleic acid(s) of interest. Largenumbers of suitable vectors and promoters are known to those of skill inthe art and are commercially available for generating the recombinantconstructs of the present invention. Appropriate cloning and expressionvectors for use with prokaryotic and eukaryotic hosts are described bySambrook et al., in Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor, N.Y. (1989), the disclosure of which ishereby incorporated herein by reference. The following vectors areprovided by way of example. Bacterial: pBs, phagescript, PsiX174,pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene);pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia). Eukaryotic:pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, andpSVL (Pharmacia). Promoter regions can be selected from any desired geneusing CAT (chloramphenicol transferase) vectors or other vectors withselectable markers. Two appropriate vectors are pKK232-8 and pCM7.Particular bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P,and trc. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, Elongation-factor-1α, early and late SV40, LTRs from retrovirus,mouse metallothionein-I, and various art-known tissue-specificpromoters. Methods well known to those skilled in the art can be used toconstruct vectors containing a polynucleotide of the invention andappropriate transcriptional/translational control signals.

Mammalian expression vectors will comprise an origin of replication, asuitable promoter and also any necessary ribosome-binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking non-transcribed sequences.Expression regulatory sequences may comprise promoters, enhancers,scaffold-attachment regions, negative regulatory elements,transcriptional initiation sites, regulatory protein binding sites orcombinations of the sequences. Alternatively, sequences which affect thestructure or stability of the RNA or protein produced may be replaced,removed, added, or otherwise modified by targeting, includingpolyadenylation signals, mRNA stability elements, splice sites, leadersequences for enhancing or modifying transport or secretion propertiesof the protein, or other sequences which alter or improve the functionor stability of protein or RNA molecules. In addition to the nucleicacid sequence encoding the diversified immunoglobulin domain, therecombinant expression vectors may carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017). For example, typically the selectable marker gene confersresistance to drugs, such as G418, hygromycin or methotrexate, on a hostcell into which the vector has been introduced. Preferred selectablemarker genes include the dihydrofolate reductase (DHFR) gene (for use indhfr⁻ host cells with methotrexate selection/amplification) and the neogene (for G418 selection).

In an exemplary system for recombinant expression of a modifiedantibody, or antigen-binding portion thereof, of the invention, arecombinant expression vectors encoding at least one antibody heavy orlight chain is introduced into dhfr⁻ CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy or light chain gene is operatively linked toenhancer/promoter regulatory elements (e.g., derived from SV40, CMV,adenovirus and the like, such as a CMV enhancer/AdMLP promoterregulatory element or an SV40 enhancer/AdMLP promoter regulatoryelement) to drive high levels of transcription of the genes. Therecombinant expression vector also carries a DHFR gene, which allows forselection of CHO cells that have been transfected with the vector usingmethotrexate selection/amplification. The selected transformant hostcells are cultured to allow for expression of the antibody heavy orlight chains. In many instances the expression vector may contain bothheavy and light chain genes, and co-transfection will lead to theproduction of intact antibody, recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium. For example, some antibodies can be isolated by affinitychromatography with a Protein A or Protein G.

The host of the present invention may also be a yeast or other fungi. Inyeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review, see, Current Protocols in MolecularBiology, Vol. 2, Ed. Ausubel et al., Greene Publish. Assoc. & WileyInterscience, Ch. 13 (1988); Grant et al., Expression and SecretionVectors for Yeast, in Methods in Enzymology, Ed. Wu & Grossman, Acad.Press, N.Y. 153:516-544 (1987); Glover, DNA Cloning, Vol. II, IRL Press,Wash., D.C., Ch. 3 (1986); Bitter, Heterologous Gene Expression inYeast, in Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y.152:673-684 (1987); and The Molecular Biology of the YeastSaccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, Vols. Iand II (1982). The host of the present invention may also be aprokaryotic organism, such as E. coli. As a representative butnonlimiting example, useful expression vectors for bacteria can comprisea selectable marker and bacterial origin of replication derived fromcommercially available plasmids comprising genetic elements of the wellknown cloning vector pBR322 (ATCC 37017). Such commercial vectorsinclude, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden) and pGEM1 (Promega, Madison, Wis., USA).

Introduction of the recombinant construct into the host cell can beeffected, for example, by calcium phosphate transfection, DEAE, dextranmediated transfection, or electroporation (L. Davis, et al., BasicMethods in Molecular Biology (1986)).

DNA encoding the antibodies of the invention is readily isolated andsequenced using conventional procedures for cloning, DNA preparation andsequencing as described by Sambrook, et al., in Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), thedisclosure of which is hereby incorporated by reference. For sequencing,oligonucleotide probes can be used that are capable of bindingspecifically to genes encoding the heavy and light chains of antibodiesor to the vector sequences surrounding the gene fragments, and the DNAsequence determined by dideoxy-based sequencing (F. Sanger, et al.(1977) PNAS 74:5463-5467). Once isolated, the DNA encoding appropriateregions of the antibody may be placed into expression vectors, which arethen transfected into host cells. The host cell can be a highereukaryotic host cell, such as a mammalian cell, a lower eukaryotic hostcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell.

In one preferred embodiment, antibodies with pairing-compatible variableregions are produced in mammalian cells. Preferred mammalian host cellsfor expressing the clone antibodies or antigen-binding fragments thereofinclude Chinese Hamster Ovary (CHO cells) (including dhfr⁻ CHO cells,described in G. Urlaub et al. (1980) PNAS 77:4216-4220), used with aDHFR selectable marker, e.g., as described in (R. J. Kaufman et al.(1982) J. Mol. Biol. 159:601-621), lymphocytic cell lines, e.g., NS0myeloma cells and SP2 cells, C127, 3T3, CHO, human epidermal A431 cells,Jurkat, U937, HL-60, mouse L-cells, Baby Hamster Kidney cells, COS orCV-1 cells, PER.C6 cells (M. G. Pau et al. (2001) Vaccine 19:2716-2721),other transformed primate cell lines, normal diploid cells, cell strainsderived from in vitro culture of primary tissue, primary explants, and acell from a transgenic animal, e.g., a transgenic mammal. For example,the cell is a mammary epithelial cell. Other cell types suitable forexpression, in particular, for transient expression, are simian COScells (Y. Gluzman (1981) Cell 23:175-182), and Human embryonic Kidneycells of lineages 293, 295T and 911 (Hek293, 295T, 911).

Alternatively, it may be possible to produce the antibody as fragment oras whole antibody in lower eukaryotes such as yeast or in prokaryotessuch as bacteria (L. C. Simmons et al. (2002) J. Immunol. Methods263:133-147). Potentially suitable yeast strains include Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida,or any yeast strain capable of expressing heterologous proteins.Potentially suitable bacterial strains include Escherichia coli,Bacillus subtilis, Salmonella typhimurium, or any bacterial straincapable of expressing heterologous proteins. If the full antibody ismade in yeast or bacteria as IgG, it may be necessary to modify theprotein produced therein, for example, by phosphorylation orglycosylation of the appropriate sites, in order to obtain thefunctional protein. Such covalent attachments may be accomplished usingknown chemical or enzymatic methods. Recombinant polypeptides andproteins produced in bacterial culture are usually isolated by initialextraction from cell pellets, followed by one or more salting-out,aqueous ion exchange or size exclusion chromatography steps. In someembodiments, the template nucleic acid also encodes a polypeptide tag,e.g., penta- or hexa-histidine. The recombinant polypeptides encoded bya library of diversity strands can then be purified using affinitychromatography. Microbial cells employed in expression of proteins canbe disrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents.

We describe here a method to directly relate the expression of the twopartner variable regions that are required to pair in such manner thatthere is minimal waste (FIG. 12). The nucleic acid encoding the firstvariable region is cloned into an expression cassette, such that it willbe under the control of a given promoter (typically the strong CMVpromoter or other), and such that its coding sequence is followed by anInternal Ribosome Entry Site (IRES) and the coding sequence of thetransactivator of the tet responsive element (TRE) fused to theactivation domain of the herpes simplex VP16 protein (tTa). The nucleicacid encoding the second variable region is cloned into an expressioncassette such that its expression is regulated via an induciblepromoter, for example, the tet responsive element (TRE), existing ofseven copies of the prokaryotic tetracycline operator site fused to aminimal CMV promoter. When introducing both expression cassettes intothe same cell (on different vectors or on same vectors, at the same timeor one before the other), the following relation between the expressionof the two variable regions will exist: expression of the first variableregion, which is under control of, for example, a constitutive promoter,will lead to the expression of the tTa protein. This protein activatesthe TRE-based promoter which will drive the expression of the secondvariable region. Thus, the production of the second variable region isnow dependent on the production of the first variable region. If theseregions are required to pair, the production of the individualcomponents of the pairing can be made dependent.

When antibodies of the IgG-type are produced via a heavy and lightchain, the production of the light chain can be made dependent on theproduction of the heavy chain. Consider the preferred embodiment, theproduction in the same host cell of a mixture of antibodies which allshare a pairing-compatible light chain. The light chain gene is clonedunder control of the TRE element, while the heavy chains are allprovided with the IRES and tTa gene, as described above. In the hostcell, every individual heavy chain that is expressed will then triggerthe production of more partner light chain. This is important, becausewith multiple heavy chains being expressed, it is likely that the levelof light chain may become limiting, and that the excess of unpairedheavy chain will induce possible toxicity in the host cell (as has beendescribed for B-cells). This concept is also applicable to theembodiment described in section 4, for the selection of antigen-reactiveantibodies from pools made in eukaryotic cells. Otherpromoter-transactivator systems have been described and are applicablein this concept also. In the same application field, in those caseswhere the ratios of two particular heavy chains need to be controlled orfixed, this method of dependent-expression may be used to link theexpression of two heavy chains.

Generally, a large number of suitable vectors and promoters are known tothose of skill in the art and are commercially available for generatingthe recombinant constructs of the present invention. The followingvectors are provided, by way of example, for the expression ineukaryotic cells of two or three antibodies that share a light chainsequence. The antibody chain encoding genes are cloned into expressioncassettes that provide all regulatory and secretion signals which aretypically used for antibody expression, as depicted in FIG. 20. In afirst embodiment, the expression of multiple antibody heavy chains ismade dependent on one another in the following way. In the firstembodiment, the nucleic acid encoding the first heavy chain (H1) iscloned into an expression cassette, such that it will be under thecontrol of a given promoter (typically the strong CMV promoter orother), and such that its coding sequence is followed by an InternalRibosome Entry Site (IRES). This is immediately followed by a secondantibody heavy chain coding region (H2, as depicted in FIG. 21). The P1promoter will now drive the expression of H1 and H2, leading to anapproximate 1:1 expression ratio between these two proteins; oftenthough the second coding region is slightly less well expressed. Thus,if the expression ratio has to be steered towards a predefined range,the use of IRES sequences is particularly useful. This predefined rangeis influenced among other factors by the nature of the IRES sequence,and different IRES sequences will mediate different final ratios.Similarly, the expression ratio between three antibody heavy chains canbe linked to one another by using a tricistronic expression cassette, inwhich the previous described cassette is followed by another IRES andHeavy chain coding region. Examples of tricistronic expression systemsand of IRES sequences and configurations are described for other systemsin the literature (Li et al., J. Virol. Methods 115:137.44; When et al.,Cancer Gene Therapy 8:361-70; Burger et al. 1999, Appl. Microbiol.Biotechnol. 52:345-53). In these embodiments, the shared antibody lightchain can be provided on a separate expression plasmid, on one or moreof the vectors that carry on or multiple the antibody heavy chains, orcan be already expressed by the host cell used for the transfection withthe heavy chain expression vector or vectors.

In another embodiment, antibody heavy genes are sequentially transfectedinto the host cell. First we consider the embodiment for libraries ofcells that produce mixes of two antibodies. Cells are transfected withthe two antibody genes cloned into different vectors but thetransfection is done sequentially in time. For example, the antibodyheavy and light chain encoding regions of the first antibody areintroduced into the host cell, and stable transfectants expressing thisantibody identified and isolated. The antibody genes encoding a secondantibody, in which the variable regions are pairing-compatible, aretransfected into the host cell that already expresses the first antibodygenes at high level. This procedure of carrying out sequentialtransfections (and if appropriate selections of integration in between)is also suitable for making collections of mixture with up to four tofive different antibodies. To increase the number of cell clonesexpressing multiple antibodies, the vectors carrying the genes encodingthe antibody genes, also carries a unique selection marker, such thattransfected cells that have integrated the vector sequence can bereadily selected and antibody expressing clones identified. As analternative embodiment for making cells that express multiple antibodieswith compatible pairing, the following procedure is used. First, asbefore, cell clone is produced that expresses one set of antibody chains(this can be one H and one L or multiple H and one L, for example) andis selected on the basis of a first selection marker. In parallel, acell clone is produced that expresses another subset of antibody chains(for example, one or more other H and one L) and that is selected on thebasis of a different selection marker (for example, neo, gpt, zeo, bdl,etc.). These cell clones are then fused and selected for the presence ofboth of the selective markers. Methods for cell fusion are extensivelydescribed in the literature and known to those working in the field;they are similar to those described in Norderhaug et al., 2002, Eur. J.Biochem. 269:3205-10. The hybrid cells have the potential to express allof the antibody chains. Similarly, this procedure can be repeated ifcollections of larger numbers of antibody chains have to be made.Further, the use of cell populations rather than cell clones, in thissequential transfection or cell-fusion approach, provides a method forachieving large collections of cells that express the antibody chains atdifferent ratios.

In one embodiment, the proteinaceous molecule's coding region or regionsare flanked by sequences that mediate site-directed integration into thehost cell genome (as depicted in FIG. 20). Without these, integration oftransgenes occurs at random and, usually, several copies of thetransgene are integrated at the same time, sometimes in the form of ahead-to-tail tandem, with the site of integration and the number ofcopies integrated varying from one transfected cell to another. The useof recombination sites as depicted in FIG. 20 allows the precise site ofintegration to be targeted by homologous recombination between vectorand host cell genome. This provides a means to insert the coding regioninto a site of high transcriptional activity, with the option to providea promoter in the transgene or use the one that is present at the siteof integration. With random or homologous recombination-mediatedinsertion of the antibody chain encoding nucleic acids is meant anyinsertion into the genome of the host cell, or into the nucleic acids ina subcellular organel, or into an artificial chromosome.

Preferred embodiments are to employ per expression vector used in thelibrary construction not more than three antibody heavy chains codingregions and preferably two per vector. Preferably plasmids do notcontain more than three promoters and three IRES sequences and not morethan six STAR or MAR elements. It is preferred to limit the expressionvector's size to 20 kb and if more binding sites than five are requiredin the mix, and these cannot be functionally encoded in a plasmid thatis less than 20 kb in size, to use two or more different plasmids.

MARs and STARs can be positioned on either side of the DNA sequence tobe transcribed. For example, the elements can be positioned about 200 byto about 1 kb, 5′ from the promoter, and at least about 1 kb to 5 kbfrom the promoter, at the 3′ end of the gene of interest. In addition,more than one element can be positioned 5′ from the promoter or at the3′ end of the transgene. For example, two or more elements can bepositioned 5′ from the promoter. The element or elements at the 3′ endof the transgene can be positioned at the 3′ end of the gene ofinterest, or at the 5′ end of a 3′ regulatory sequence, e.g., a 3′untranslated region (UTR) or a 3′ flanking sequence. Chromatin openingelements can be flanking on both ends of the expression cassette (FIG.21D), or placed 5′ of the expression cassette (FIG. 21C). In particular,when multiple regulatory elements such as STAR and UCOs have to beintroduced into one and the same plasmids, preferably elements are usedthat have activity towards both ends of the element such that they canbe provided in the middle of an expression cassette (FIG. 21C). SinceMARs have also been reported to function when co-transfected in transwith the transgene (Zahn-Zabel et al. (2001) J. Biotechnology 87:29-42),they have the advantage that no DNA-cloning step is required tophysically link them to SPCBP expression cassette(s). In that case sizeof the MAR element or of the expression vector carrying the SPCBPcassettes is no longer a limitation. Nevertheless, MAR elements as smallas 1.3 kb have been described, thus multiple in cis inclusions arefeasible. MARs have also been reported to be added both in cis and intrans, and in this configuration increase expression levels ofantibodies in CHO cells 14-fold. One other function of these elementsbesides their effect on stability is that they will also increase thenumber of independently transformed cells that express the protein andpromotes higher amounts of the recombinant protein. Clone isolation andproduction levels are overall higher, thus in a preferred embodiment,this invention is practiced by using these elements for making largecollections of cell lines producing compositions comprising multiplefunctional binding sites.

8. Proteinaceous Mixtures with Multiple Effector Regions and MultipleTypes of Binding Sites

The invention can be used to create compositions of proteinaceousmolecules that have multiple effector regions. In the case ofantibodies, compositions are included that display one or more antigenbinding regions in combination with two or more natural effectorregions. Examples are the effector regions encoded by IgG1 and IgG4,which have, for example, different binding regions for C1q and thevarious Fc-receptors based within their encoding constant regions. Suchmixtures may be clinically more effective than their mono-effectorcompounds: the mixture combines multiple and maximal natural effectors,which for various reasons are never present in the one natural antibodyisotype, and the mixture thus mimics much more closely the naturalpleiotropy of immune effectors that a single antigen/pathogen will evokewhen our immune system encounters it. Some formats are IgG1 and IgG4, orIgG and IgM, or IgG1 and Fab, or IgG and IgA, or IgA and IgM, orIgG1-cytokine fusion and alike. Instead of making such proteins indifferent hosts, the co-expression of such different antibody formats,all associated with the same binding site (or possibly multiple bindingsites but related to one target and preferable to one disease orindication), allows the direct production of cocktails of antibodieswith different effectors. Such mixtures are more efficacious in theirbiological activity.

Besides antibodies, recent protein engineering techniques have allowedthe production of binding sites with predetermined specificity usingsimilar but also sometimes using very different structures. For example,antigen-specific ligands have been created using phage, bacterial,ribosomal or yeast display methods, from libraries of protein variants,in which the protein at some positions was variegated using random oroligonucleotide-based mutagenesis, but the main scaffold of the nativeprotein maintained in the variants. Proteins for which has been alreadyapplied include the protein Z domain of Protein A, a variety of Kunitzdomains, lipocalins, Green Fluorescent protein, one of the fibronectindomains, other domains of the immunoglobulin superfamily, and ankryns.Such antibody mimics are thus proteinaceous molecules with a non-naturalbinding activity, obtained, for example, by engineering into themolecule one or more residues or regions with variegated sequences, ateither defined or random positions, and identifying the molecule withappropriate antigen binding properties by screening or selectionprocesses. Examples of the processes are high-throughput screening forantigen binding by ELISA, or selection methods described in theliterature such as in vitro display methods such as ribosome andpuromycin display, cellular or viral display methods such as filamentousphage, lambda phage, bacterial, yeast, or eukaryotic cell display. Theresulting proteinaceous molecules with the new binding site is anantibody mimic in the sense that it will contain a binding region forantigen at the position where it was initially a variable region,similar to an antibody molecule with two variable regions.

9. Making Compositions of Multiple Proteinaceous Compounds withDifferent Binding Specificities.

Recombinant DNA technology provides methods well known in the art toclone the variable region genes, and produce cell lines expressing therecombinant form of the antibody. In particular, the properties ofantibodies are being exploited in order to design agents that bind tohuman target molecules, so-called “self-antigens,” and to antigens ofviral or bacterial diseases. For example, a number of monospecificantibodies have been approved as human therapeutics. These includeOrthoclone OKT3, which targets CD3 antigen; ReoPro, which targetsglycoprotein IIb/IIIa; Rituxan, which targets CD20; Zenapax andSimulect, which target interleukin-2 receptors; Herceptin, which targetsthe HER2-receptor; Remicade and Humira, which target tumor necrosisfactor; Synagis, which targets the F protein of respiratory syncytialvirus; Mylotarg, which targets CD33; and Campath, which targets CD52.

For many clinical applications the efficacy of the treatment wouldincrease if combinations of monoclonal antibodies are used. Anoligoclonal preparation can be made by mixing individual recombinantantibodies which each have been made by conventional procedures, whichincludes the expression and purification of the individual recombinantor hybridoma-derived monoclonal antibodies, and the subsequent mixing ofthese molecules. The pharmaceutical development of separately producedand then mixed monoclonal antibodies is inhibitively expensive.Recombinant monoclonal antibodies of the IgG isotype are commonly madeby co-expression of the nucleic acid sequences encoding the heavy andlight chain of the antibody in the same host cell, yielding a monoclonalantibody, bearing two identical binding sites. The production of severalantibodies from individual cell lines each making one antibody (and inwhich each cell line is controlled for stability of expression andconsistency), is not economical with present biotechnological productionmethods.

One approach to combine monoclonals is to combine the binding sites inone molecule, hence creating a multispecific antibody. This allows thetargeting of multiple epitopes on the same antigen, or of multipleantigens on the same target entity (e.g., a cell, a virus, a bacteria,an antigen), or of epitopes on different entities, providing a bridgebetween these entities. Of the multispecific antibodies, bispecificantibodies have been investigated the most, for targeting therapeutic ordiagnostic entities to tumor cells, e.g., a cytotoxic T-cell, an NKcell, a chelator that carries a radionuclide. But in the bispecificantibody the two binding sites are always covalently coupled to oneanother, which limits the flexibility and use of such compounds.Further, many of the recombinant bispecific antibodies (e.g., Fab-scFvfusions, diabodies, double-single-chain Fvs) lack the provision of theantibody's Fc region. Since Fc-dependent effector mechanisms such asADCC are important for the efficacy of many antibodies (e.g., Rituxanand Herceptin), it will be important to maintain this region in themultispecific molecule.

The alternative approach is to use polyclonal antibodies comprising theentire immune response of a host to an immunogen. Polyclonals derivedfrom the pooled serum from immunized animals or from selected humanshave been used therapeutically e.g., for passive or active immunization,e.g., anti-rhesus D, anti-digoxin, anti-rabies, anti-snake venompolyclonals, and in some instances, work more effectively than acomparable monoclonal, e.g., Sangstat's rabbit polyclonal againstthymocytes versus Simulect™. Drawbacks for the use of polyclonalantibodies are the risk of infectious agents (viruses, prions, bacteria)in these often pooled preparations, but also the variability inefficacy, the limited availability, the immune response directed to thepreparation if the polyclonal is non-human, and the abundance ofnon-relevant antibodies in these preparations. Polyclonals have alsobeen made using recombinant methods, but again, the production of largearrays of antibodies from individual cell lines each making oneantibody, is not economical with present biotechnological productionmethods. The production of the polyclonal antibody mixture bycultivating the many different cell lines in batch would be even moreaffected by differences in stability, growth and production rate,differences in purification yield, etc.

The present invention provides methods to produce mixtures ofantibodies, preferably by expression from a single host cell, usingantibodies with variable regions that appropriately pair with oneanother to yield essentially solely functional binding sitecombinations. The methods to obtain such antibodies were describedearlier. The resulting variable regions can thus be co-expressed inbiotechnologically viable and simple procedure, and a mixture ofantibodies isolated using methods known in the art.

After selection of antibodies with the appropriate pairing behavior(such as antibodies with pairing-compatible variable regions,co-expression-compatible elements, etc., as described above), theantibody variable region genes are cloned into expression vectors thatwill direct the expression of an antigen binding fragment in, forexample, the following format: Fab, Fab′, Fab′2, IgG, IgM. In manyinstances the use of antibodies with, for example, pairing-compatiblevariable regions simplifies the DNA constructs that mediate theexpression of the particular antibody format. For example, for theexpression of two different antibodies as Fab′2 fragments in which oneof the two antibody chains is the pairing-compatible variable region,only three antibody chains instead of the normal four have to beexpressed to form two different binding sites. Such simplifiedexpression constructs can lead to a more stable and more readilycontrolled expression system, and increase functional yields byminimizing problems associated with mispairing of heavy and light chaindomains.

The mixture may contain a given selection of antibodies, recognizingepitopes on the same or different targets; examples are given below. Anew application is the use of mixture containing antibodies specific forcomplexes formed by another antibody bound to a given target. Both ofthe antibodies can be provided in the mixture, providing a firstantibody to bind the antigen, and a second one to “seal” the firstinteraction, providing the antibody mixture with an increase in overallaffinity and specificity. Another embodiment of the invention is to useasymmetrically paired antibody molecules in the mixture such that theeffector functions of the resulting mix are altered. The purpose of suchmixing is to alter the properties of the effector mechanism of theindividual antibodies in the mixture, in an antigen-specific/bindingsite directed manner, for example, the monospecific antibodies may eachhave a different effector from the bispecific components present in themixture. Consider the next example, a mixture of two antibody bindingsites formatted as Oligoclonics™ in the IgG-format, composed of theheavy chain gamma-1 heavy chain for one antibody variable region and thegamma-4 heavy chain for the other antibody variable region. TheOligoclonics™ mixture will contain the two monospecific antibodies,which will be either an IgG1 or an IgG4 isotype and display theirrespective effector functions, and also a hybrid dimer of gamma-1 andgamma-4, with altered or lost effector functions. Since many Fcreceptors bind in an asymmetric manner to the symmetrically arranged Fcregion, asymmetric Fc regions often will loose interactions with Fcreceptors and thus ADCC or other activity. Mutants of Fc regions with,for example, mutations in the Fcgamma-Receptor motif (residues 233-238in the CH2-lower hinge region), or mutants with reduced C1q binding, ormutants with exchanged or shortened hinge, or with domains exchanged byother domains of the immunoglobulin heavy chain family, or Fc regionsoptimized for their interaction with particular Fc regions (e.g.,improved binding to the activating receptor FcgammaRIII and/or decreasedbinding to the inhibitory receptor FcgammaRIIb), can also be used forthe assembly of such asymmetric Fc regions. Applications of suchasymmetric pairs are provided in a mixture of one compound but notothers with a particular effector function, or to remove an effector,for example, in the bispecific or monospecific compounds.

10. Examples of Uses of Compositions of Multiple Proteinaceous Compoundswith Different Binding Specificities

There are applications for mixtures of different binding sites on thesame antigen, for mixtures of different binding sites on differentantigens, for mixtures of different binding sites on different antigenson the same or different target. As an example of use of a mixture inthe treatment of a viral disease, the example of Hepatitis B virus (HBV)infection is discussed. Recombinant HBV vaccines provide a safe andeffective means for prevention of HBV conferring long-term immunitythrough active immunization. In contrast to the slow onset of protectionfollowing this vaccination, passive immunotherapy with antibodies to HBVprovides immediate but short-term protection against viral transmissionand infection. Antibodies are believed to inhibit infection by blockingHBV from entering into cells. Such passive immunotherapy is advisablefor individuals who were exposed to HBV-positive material (needle or cutinjuries) and for newborns to mothers who are HBV carriers, for patientsundergoing liver transplantation. At present, such treatment is carriedout with Hepatitis B immunoglobulin, a plasma derived, polyclonalantibody preparation obtained from donors who were anti-hepatitis Bsurface antigen antibody-positive. The availability of this serum islimited and further pricing and safety concerns regarding the use ofblood products, make the development of an alternative treatmentnecessary. A human monoclonal antibody would be advantageous bypresenting a stable and reproducible source for prolonged immunotherapy.However, studies show that a monoclonal antibody directed to the Santigen and neutralizing capacity against HBV in chimpanzees delayed butnot prevented the infection with HBV. In part this may be caused by theemergence of escape variants, mutants in the S-antigen that can nolonger be bound by the monoclonal antibody. Similarly, escape mutantsarise in patients after liver transplantation in clinical trials withmonoclonal antibodies. Therefore, treatment with a single monoclonalantibody may be inefficacious and insufficient. Follow-up studies haveinvolved mixtures of human monoclonal antibodies. Studies carried out byXTL Biopharmaceuticals and colleagues show that a mixture of twoantibodies is more effective in reducing viral load and inhibiting HBVinfection in animal model systems than a polyclonal mixture. Thisindicates that the potency of a polyclonal humoral immune response canbe deconvoluted to a few antibodies, and that a defined mixture of a fewantibodies should work as well or better than some polyclonalpreparations. A mixture of two antibodies recognizing different epitopeson the viral surface was thus shown to be more effective in theprevention of HBV reinfection.

In another example of use of a mixture of monoclonal antibodies in thetreatment of a viral disease, the example of the Human ImmunodeficiencyVirus type-1 (HIV-1) is discussed. Infection with HIV-1 will lead to thedevelopment of the Acquired Immunodeficiency Syndrome (AIDS) if leftuntreated. During infection with HIV-1, neutralizing antibodies that aredirected against diverse epitopes on the HIV-1 envelope glycoproteinmolecules gp41 and gp120 develop. In a clinical trial published in 1992,the administration of HW-1 seropositive plasma containing high titers ofHIV-neutralizing antibodies, was associated with a reduction in HIV-1viremia and a number of opportunistic infections. Several groups havesubsequently published that administration of HIV-1 seropositive plasmaresults in delay of the first AIDS-defining event and improvement ofclinical symptoms. However, enthusiasm for passive immunotherapydeclined when it was found that antibodies failed to eliminate the virusand resulted in the emergence of neutralization escape variants inpatients. It was demonstrated that the antibodies that are inducedduring natural HIV-1 infection poorly neutralize the virus, resulting ina low potency of hyperimmune sera used for passive immunotherapy ofHIV-1 infection. In addition, it was demonstrated that some antibodiesthat arise during natural infection can even enhance the infection. Itwas realized that for antibody therapy of HIV-1, potent andwell-characterized neutralizing monoclonal antibodies were needed.

These early findings spurred the development of human monoclonalantibodies against HIV-1 envelope glycoproteins. In recent years, anumber of human monoclonal antibodies against the HIV-1 gp41 and gp120viral coat glycoproteins have been isolated and characterized for theirvirus-neutralizing activity in vitro. Subsequent experiments innon-human primate models of HIV infection and transmission have shownthat human monoclonal antibodies targeting different HIV-1 envelopeglycoprotein epitopes exhibit strong synergy when used in combination.It has been suggested that combinations of human anti-HIV monoclonalantibodies can be exploited clinically for passive immunoprophylaxisagainst HIV-1.

The third example relates to the Rabies field. Rabies is an acute,neurological disease caused by the infection of the central nervoussystem with rabies virus, a member of the Lyssavirus genus of the familyof Rhabdoviridae. Almost invariably fatal once clinical symptoms appear,rabies virus continues to be an important threat to human and veterinaryinfection because of the extensive reservoirs in diverse species ofwildlife. Throughout the world, distinct variants of rabies virus areendemic, in particular, terrestrial animal species, with relativelylittle in common between them. Rabies virus is characteristicallybullet-shaped, enveloped virion of single-stranded-negative sense RNAgenome and five structural proteins. Of these, a suitable target forneutralization is the viral glycoprotein (G). Antigenic determinants onG vary substantially among the rabies virus strains. Prompt treatmentafter infection consists of passive and active immunotherapy. Forpassive immunotherapy mostly pooled serum of rabies immune individualsor immunized horses is used, but with a risk of contamination with knownor unknown human pathogens, or the risk of anaphylactic reactions,respectively. In addition, anti-rabies immunoglobulin is expensive andmay be either in short supply or non-existent in most developingcountries where canine rabies is endemic. There is, therefore, a needfor compositions and methods for producing mixes of antibodies,preferably human antibodies, to use in passive immunotherapy of Rabiesinfections. A number of human monoclonal antibodies made by fusion ofEpstein-Barr Virus transformed, rabies-virus-specific humanheterohybridomas have been made (Champion et al., J. Immunol. Methods(2000) 235:81-90). A number of virus-neutralizing antibodies derivedfrom these antibodies have also been cloned (PCT/IS02/26584 andPCR/US01/14468 and Morimoto et al. (2001), J. Immunol. Methods252:199-206). Several other rabies-neutralizing monoclonal antibodieshave been described in the art, which could also be used in theexperiments below. As indicated in these publications, a mix ofdifferent rabies-neutralizing human antibodies would be an ideal reagentfor passive immunotherapy of Rabies.

In general for viral diseases, the functional assembly of mixes ofanti-viral antibodies may increase the clinical efficacy of thetreatment when compared to monoclonal therapy, by decreasing theprobability of viral escape mutants resistant to treatment, and byreducing the likelihood of viral resistance with prolonged therapy. Inthe mixture, antibodies may be included that bind to many differentepitopes of the virus. It may also be feasible to include antibodies todifferent subtypes of the virus, to broaden the utility of the drug fora wider patient population. Further anti-viral antibodies directed tolinear epitopes may be added, which may be less prone to the effect ofescape mutants than conformation-dependent antibodies. The effect ofmultiple binding specificities present in the antibody mix can provide astronger signal for viral clearance than when a monoclonal antibody isused. There are also applications for mixtures of essentially onebinding site with different fine-specificities for binding its antigen.For example, when the antigen is prone to mutation as is the case withmany viral antigens, in the course of a treatment the epitope on theantigen may be altered such that the binding of the original antibody islost. When using a mixture, e.g., based on the same heavy chain pairedwith a small set of light chains that provide a range offine-specificities, there is a possibility that the mutations willaffect the binding of some species in the mixture, but not of otherswith a different binding chemistry mediated by the pairing-compatiblevariable region. In such a case it will be preferable to use distinctbinding chemistries for the interaction with the antigen, thus thepairing-compatible variable regions should be as unrelated as possiblein sequence. Alternatively, antibodies can be used that use verydifferent binding site chemistries by having unrelated heavy and lightchain variable regions, but display exclusively pairing behavior suchthat their production in the same cell yields mainly functional bindingsites. Such mixtures are preferably more active than the individualcomponents, and in some case will act synergistically.

In the Oligoclonics™ format, antibodies of the IgG isotype are made byco-expression of the light and heavy chain genes with appropriatepairing behavior in the same host cell. The result of this process is amixture of different proteins, the monospecific bivalent antibodieswhich carry two identical binding sites, and bispecific antibodies,carrying two different binding sites. There will be occasions where thepresence of this bispecific antibody class will further enhance theefficacy of the antibody mixture. Only when there are multiple epitopespresent on the antigen or microorganism, and these epitopes arepresented in the correct positioning, will a monoclonal antibody of theIgG isotype, for example, be able to bind both of its binding Fab-armsto the antigen. In many instances where the antigen is a monomer or asmall multimer, like cytokines, interleukins and interferons, mostlyonly one arm of a monoclonal IgG antibody will be binding the antigen.The bispecific component of the Oligoclonics™, provides a newopportunity to bridge neighboring epitopes, and form a highly avidbinding antibody. Pairs that have this behavior may be selected usingthe methodologies of screening mixtures of antibodies as disclosedherein. Besides this avidity advantage, bispecific molecules may alsocross-link receptors that mono-specific yet bivalent antibodies in thesame mixture cannot cross-link. Oligoclonics™ may thus provide anantibody mixture that per unit of mass will more effectively neutralizeviruses, cytokines, toxins etc when compared to monoclonal antibodies,and in specific cases, for example, with an avidly binding bispecificcomponent or receptor-cross-linking or other unique mechanism mediatedby the bispecific antibody, also compared to mixtures of monoclonalantibodies. The bispecific compounds are also useful to explore routestraditionally developed with bispecific antibodies, such as theretargeting of immune effector molecules or cells such as T-cells,complement proteins and Fc-receptor expressing cells to tumor cells orpathogens.

Thus, mixtures of antibodies may be suitable to fight pathogensincluding viruses like HIV and Rabies, bacteria, fungi and parasites.Other examples where a polyclonal serum or gammaglobulin is currentlyused that could be replaced with a defined antibody mixture, includesuch diseases as rabies, hepatitis, varicella-zoster virus, herpes orrubella. Bacterial diseases that could be treated with antibody mixturesinclude Meningitis, diseases caused by Staphylococcus, Streptococcus,Hemophilus, Nesseria, Pseudomonas and the actinomycetes. Targets mayalso include those involved in bacterial sepsis such aslipopolysaccharide (LPS), lipid A, tumor necrosis factor alpha orLPS-binding proteins. Some of these pathogens occur in multipleserotypes and not one but multiple antibodies are required to neutralizethe various serotypes. A mixture of antibodies will provide, by thechoice of the binding specificities, a wider coverage of serotypes thatmay be treated and, therefore, more patients can be treated with thesame antibody mixture. The mixtures for this and other reason can formalso suitable diagnostics and part of diagnostic kits for the detectionof a disease or disorder in patient.

Mixtures of antibodies may be more effective than monoclonal antibodiesalso in the treatment of oncological diseases such as non-Hodgkin'slymphoma (NHL) and epithelial cell tumors like breast and coloncarcinoma. Targeting both CD20 and CD22 on NHL with antibodies hasalready been proven to be more effective than targeting the tumor cellswith the individual antibodies. Suitable target antigens for antibodymixtures in oncological diseases are many, including CD19, CD20, CD22,CD25 (IL-2 receptor), CD33, the IL-4 receptor, EGF-receptor, mutant EGFreceptor, Carcino-Embryonic Antigen, Prostate-specificAntigen,ErbB2IHER2, Lewis^(y) carbohydrate, Mesothelin, Mucin-1, the transferrinreceptor, Prostate-specificMembrane Antigen, VEGF and receptors, EpCAMand CTLA-4. Synergistic effects may be seen when using antibodies thatbind different targets and pathways in the disease, such as antibodieswith anti-angiogenesis and anti-proliferative effects. There are alsoapplications in this field for a mixture of essentially one binding sitewith different affinities for binding its antigen. For example, theefficiency of in vivo solid tumor penetration is limited for highaffinity antibodies due to the binding site barrier, yet a minimalaffinity is required to achieve a substantial accumulation in the tumor.With the methods described in this document, a mixture of antibodies canbe established, e.g., based on the same heavy chain paired with a smallset of light chains yet appropriate pairing behavior that provide arange of affinities when paired with the heavy chain. Such mixtures canbe used to increase the accumulation in the tumor, and the best balancedcocktail found by choosing the components and their expression levels.Such mixtures are preferably more active than the individual components,and may act synergistically.

Mixtures of antibodies may also be suitable to neutralize multipledifferent targets, for example, in the field of inflammatory diseases,where multiple factors are involved one way or another in mediating thedisease or aggravating its symptoms. Examples of these diseases arerheumatoid arthritis, Crohn's disease, multiple sclerosis,insulin-dependent diabetes, mellitus and psoriasis. Optimal treatment ofmany of these diseases involves the neutralization or inhibition ofcirculating pathological agents and/or those on the surface on cellstargeted in the specific inflammatory response in the patient. Inautoimmunity and inflammatory diseases suitable targets are generallyinterferons, cytokines, interleukins, chemokines and specific markers oncells of the immune system, and, in particular, alpha interferon, alphainterferon receptor, gamma interferon, gamma interferon receptor, tumornecrosis factor alpha, tumor necrosis factor receptor, HLA-class IIantigen receptor, interleukin-1beta, interleukin-1beta receptor,interleukin-6, interleukin-6 receptor, interleukin-15, interleukin-15receptor, IgE or its receptor, CD4, CD2, and ICAM-1.

Mixtures are also suitable for the neutralization of effects mediated byagents of biological warfare, including toxins such as Clostridiumbotulinum derived botulinum neurotoxin, Anthrax, smallpox, hemorrhagicfever viruses and the plague. The neutralization of the botulinum toxinsis discussed here as an example. The botulinum toxins, the mostpoisonous substances known, cause the paralytic human disease botulismand are one of the high-risk threat agents of bioterrorism.Toxin-neutralizing antibody can be used for pre- or post-exposureprophylaxis or for treatment. Small quantities of both equine antitoxinand human botulinum immune globulin exist and are currently used totreat adult and infant botulism. Recombinant monoclonal antibody couldprovide an unlimited supply of antitoxin free of infectious disease riskand not requiring human donors for plasmapheresis. A panel of human andmurine monoclonal antibodies was generated from the B lymphocytes ofhyperimmune donors and immunized mice using phage antibody displaytechnology. Single monoclonal antibodies and combinations were testedfor their capacity to protect mice from lethal doses of neurotoxin (A.Nowakowski et al. (2002) PNAS 99:11346-11350.). Whereas singlemonoclonal antibodies showed no significant protection of the miceagainst lethal doses of toxin, combinations of only three monoclonalantibodies against different epitopes on the toxin gave very potentprotection. The combination of three monoclonal antibodies neutralized450,000 lethal doses of botulinum toxin, a potency 90 times greater thenhuman hyperimmune globulin. Importantly, the potency of the monoclonalantibody mixture was primarily due to a large increase in functionalantibody-binding affinity. Thus, methods that allow the cost-effective,controlled and efficient production of mixtures of monoclonal antibodiesagainst botulinum neurotoxin provide a route to the treatment andprevention of botulism and other pathogens and biologic threat agents.As shown in this study, a mix of three antibodies that boundnon-overlapping epitopes on botulinum neurotoxin, had a synergisticeffect on toxin neutralization due to a increased overall avidity.

Mixtures of antibodies may be further applied to delay the onset ofanti-idiotype responses in patients, by providing multiple idiotypes ofan antibody family, all binding to the same target, in the simplest formamino acid mutants of the same antibody with a resulting similar bindingspecificity and affinity, to a more complex mixture of multipleantibodies directed to the same epitope.

Mixtures of antibodies can also be applied to develop derivatives of theprotein mixtures, including immunotoxins, immunoliposomes, radio-isotopelabeled versions, immunoconjugates, antibody-enzyme conjugates forprodrug-therapy (ADEPT), and immunopolymers (Allen, (2002) Nat. Rev.Cancer 2:750-783). The mixes of the antibodies can either be modified inbatch with the appropriate substances, or may be genetically fused to atoxin or enzyme encoding gene as described in the art for monoclonalantibodies.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Description of the Hybridoma-Derived Anti-RabiesAntibodies Used in the Studies

This example describes a number of rabies-neutralizing antibodies thatare used in the further examples. The following antibodies arevirus-neutralizing human antibodies: (1) JB1 (abbreviated to JB in thenext section), described in Champion et al., J. Immunol. Methods (2000)235:81-90, and the cloning and sequence in PCT/IS02/26584; (2) JA-3.3A5(abbreviated to JA in the next section), described in Champion et al.,J. Immunol. Methods (2000) 235:81-90, the cloning in Morimoto et al.(2001), J. Immunol. Methods 252:199-206 and also in PCT/US01/14468; (3)M57, antibody and its cloning were described in Cheung et al. (1992), J.Virol. 66:6714-6720, and further in PCT/IS02/26584. The nucleotidesequences of the full heavy and light chain nucleotide sequences andalso amino acid sequences of their variable regions are disclosed in thesequence listings (SEQ ID NOS:1-12). On the basis of the data in theliterature these antibodies all neutralize a variety or rabies isolates,but not all the same, providing a broader spectrum of neutralizedisolates than when using a monoclonal.

Example 2 Production of Mixtures of scFv Antibody Fragments Based onRecloned Hybridoma-Derived Anti-Rabies Antibodies and Co-Expression

This example describes the production of a mixture of three bindingsites as three proteins. Using as template the variable region genes ofthe three antibodies described in Example 1, cloning is used toconstruct three single-chain Fv expression cassettes, one for each ofthe antibodies, and to clone these in an appropriate expression vector.

First the variable region genes are amplified with oligonucleotides thathybridize to the 5′ and 3′ ends of the nucleotide sequences and provideappropriate restriction enzyme sites for cloning. Standard cloningtechniques are described in Sambrook et al., Molecular cloning, secondedition, Cold Spring Harbor Laboratory Press (1987). Cloned variableregions genes are amplified by the polymerase chain reaction usingmethods well known in the art. For antibody JA, the following procedureis used: primers are designed in the FR1 region and in the FR4 region ofthe variable heavy chain nucleotide sequence, such that the variableregion is cloned downstream of a bacterial leader sequence and upstreamof a continuation of the reading frame with a Gly-Ser encoding sequence.The polylinker into which the variable region heavy and light chains arecloned is indicated in FIG. 13. The primers are designed to maintain theamino-terminal sequence of the FR1 and FR4 regions, and to include aunique restriction enzyme site for cloning of the variable region intothe polylinker region of pSCFV (FIG. 13). pSCFV is a pUC119 derivatewhich is essentially pHEN 1 (Hoogenboom et al. (1991) Nucl. Acids Res.19:4133-4137) into which the SfiI-NotI fragment is replaced with theSfiI-NotI sequence depicted in FIG. 13, and in which the NotI site isfollowed by a c-myc tag, for detection and purification of the antibodyfragment. Also the geneIII of filamentous phage is deleted in thisplasmid. Several options for directional cloning are feasible, indicatedby the restriction sites locations on the polylinker map on FIG. 13. Forthe VH of JA, the following oligonucleotides are used to amplify the VHregions: 5′-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCA GAG GTG CAG CTGTTG GAG TCT GGG GG-3′ (SEQ ID NO:18) and the reverse complement of5′-ACC CGG GTC ACC GTC TCC TCC-3′ (SEQ ID NO:19). The PCR reaction iscarried out with the template antibody gene which was already cloned,plasmid SPBN-H (Morimoto et al. (2001), J. Immunol. Methods252:199-206), for 25 cycles, denaturation at 94° C. for 30 seconds,annealing at 50° C. for 60 seconds, and elongation at 72° C. for 90seconds, using Taq DNA polymerase (Promega, Madison, Wis.). Theresulting product of approximately 400 by is purified, digested with therestriction enzymes SfiI and BstEII, and cloned into pSCFV, resulting inpJA-VH. Similarly, the light chain of JA is amplified from pSPBN-L withappropriately designed oligonucleotides and is cloned into pJA-VH, toyield pSCFV-JA. The integrity of the sequences is confirmed by using theAmpliTaqs cycle sequencing kit (Perkin-Elmer, Foster City, US) withspecific primers based in the vector backbone just adjacent to thevariable region inserts. Similarly, the antibody variable regions fromhybridomas JB and M57 are cloned into the single-chain Fv format.

The expression of the individual antibody fragments is done as follows.Soluble scFv fragments are expressed upon induction withisopropyl-β-D-thiogalactopyranoside (IPTG) from the lacZ promoter thatdrives the expression of the scFv in pSCFV-based plasmids, and harvestedfrom the periplasmic space of E. coli TG1 cells. To confirm binding ofthe individual scFvs, an ELISA is performed using Polysorb plates (Nunc)coated with 5 micrograms/ml of rabies virus glycoprotein. Viruspurification and glycoprotein purification have been described elsewhere(Dietzschold et al. (1996) Laboratory Techniques in Rabies, Eds Meslin,Kaplan and Korpowski), World Health Organization, Geneva, p. 175).Alternatively, a source of recombinant Rabies Glycoprotein (G) of theappropriate type is used for the coating. The sequence of rabies G isavailable to the person skilled in the art and so are cloning,expression and purification techniques.

In the next step, the scFv expression cassettes are cloned one afteranother in plasmid pSCFV-3 (depicted in FIG. 14A), which is a derivativeof pSCFV carrying unique restriction sites for cloning scFv genes, twobehind the same lacZ promoter and separated via a new ribosome-bindingsite (rbs) and signal sequence (S), and one behind anarabinose-inducible promoter, rbs and S (FIG. 14A). It also carriesdifferent tags, one for each of the scFv cassettes, c-myc (as in pSCFV;sequence EQKLISEEDL (SEQ ID NO:20)), the VSV-tag (the sequenceYTDIEMNRLGK (SEQ ID NO:21)) and the influenza Hemagglutinin (HA)-tag(the sequence YPYDVPDYA (SEQ ID NO:22)), and all followed by a stretchof three alanines and five histidines. This set-up provides a method fordetection of the individual antibodies in the mix, and a generic methodfor purification, based on immobilized metal affinity chromatography(IMAC) using methods well known in the art. The plasmid is also used inExample 17 (with restriction inserts and cloning sites described in SEQID NOS:16 and 17). The scFv genes are amplified with oligonucleotidesthat introduce the appropriate sites, and cloned into this plasmid.

The finally resulting plasmid, pSCFV-JA-JB-M57 (FIG. 14B) is introducedinto E. coli host TG1 cell, and expression of the scFvs induced withIPTG (for JA and JB) and/or arabinose (M57). By induction with IPTG, theexpression of a mixture of two functional scFv fragments is achieved, inwhich the direct linkage favors the pairing between the intramolecularlylinked variable regions. By further induction with arabinose, anadditional scFv fragment is co-expressed. Alternatively, the three scFvexpression cassettes are cloned in separate plasmids into compatibleplasmids such as pBR322 and pACYC and maintained in the same host cellbefore induction. The binding of the mixture to rabies glycoprotein (G)is tested as before using ELISA. The contribution to the binding in themix of each of the scFv fragments is confirmed using one of threeanti-tag antibodies (the mouse monoclonal antibody 9E10 binding to humanc-Myc epitope tag (product code from abcam, www.abcam.com: ab32), andpolyclonal antibodies to the HA-tag (ab3413) or VSV-tag (ab3556). Toverify whether the production is carried out by one bacterium and itsprogeny and not by three clones that each produce one of the antibodyfragments, the culture is colony-purified after four hours in theinduction phase and the production tested of three independent clones,confirming that the expression is clonal. To determine the percentage ofcorrectly paired variable regions, the scFv mixture is first purifiedfrom the E. coli periplasmic extract using IMAC. Briefly, an IPTG andarabinose-induced 500 ml culture (kept for four hours at 30° C.), isspun at 4600×g for 20 minutes at 4° C., and the bacterial pelletresuspended in phosphate buffered saline (PBS) containing proteaseinhibitors (phenyl-methyl-sulfonyl fluoride and benzamidin). Thesolution is sonicated at 24° C. using an ultrasonic desintegrator (MSEScientific Instruments), and the suspension centrifuged at 50,000×g for30 minutes at 4° C. The supernatant fraction is incubated with TALON™resin according to the instructions of the manufacturer (Clontech).After extensive washing, proteins are eluted using 100 mM imidazole.Following this procedure, scFv fragments are further purified by gelfiltration using a Superdex 75 column (Amersham Pharmacia Biotech)connected to a Biologic instrument (Biorad). ScFv concentrations arequantitated using the bicinchoninic acid kit (Pierce). A fraction of theantibody mix is bound to a molar excess of biotinylated G protein in a0.5 ml volume. The protein with bound scFvs is captured onto the surfaceof an excess of paramagnetic Streptavidin-coated beads (200 microlitersof DYNAbeads, Dynal, Norway), in a way similar to what is described inExample 4 for phage selections. The supernatants of the mixture are thentested for the presence of scFv fragments in an SDS PAGE followed byWestern blot analysis with the anti-tag antibodies to characterize thenon-functional antibodies. The experiment provides evidence for thesimultaneous production of three scFv fragments by the same host cell,and the efficient recovery of functional binding sites, thus correctlypaired variable regions from this preparation.

Example 3 Production of Mixtures of scFv-Fc Antibodies Based on ReclonedHybridoma-Derived Anti-Rabies Antibodies and Co-Expression in aEukaryotic System

This example describes the production of a mixture of three or sixdifferent proteins composed of variable regions paired to form two orthree binding specificities. In a further example, the scFv genes aresubcloned into a eukaryotic expression vector based on pcDNA3 whichcarries the human gamma-1 region. This plasmid, VHExpress, wasextensively engineered to remove internal restriction enzyme sites(Persic et al. (1997) 187:9-18), and contains a promoter (CMV instead ofEF-1alpha as in publication), a eukaryotic leader sequence, a polylinkerwith cloning sites for an antibody variable region, the human gamma-1gene and the bovine growth hormone poly A site (FIG. 15). Further itcontains the genes encoding amp and neo resistance, and the SV40 originof replication. The full sequence is given in SEQ ID NO:13. This vectoris suitable for the expression of antibody variable region genesformatted as scFv fragments. The cloning of the scFv gene of antibody JAis carried out as follows. The scFv is used as a template in a PCRreaction with oligonucleotides 5′-TATC CGC GCG CAC TCC GAG GTG CAG CTGTTG GAG TCT GGG GG-3′ (SEQ ID NO:23) and the reverse complement of5′-ACC CGG GTC ACC GTC TCC TCC GGT GAG TCC TAG CGC TTT TCG T-3′ (SEQ IDNO:24). The PCR fragment of approximately 750-800 by is isolated,digested with BssHII and Eco47III and cloned into similarly cut plasmidVHExpress. Similarly, the scFv genes of antibodies JB and M57 are clonedinto this plasmid; to avoid digestion at internal sites the othersuitable site is used (Bpu1102I) or a three-way ligation which alsoyields the same plasmid. The resulting plasmids with correctly clonedscFv, called respectively pscFv-Fc-JA, pscFv-Fc-JB and pscFv-Fc-M57, areintroduced into host cells, in this example PER.C6™ cells.

For an initial analysis, these plasmids are transiently expressed eitheralone or in combinations of two or three scFv-Fc constructs. Cells grownto 5×10⁶ cells/ml in culture medium with 10% Fetal calf serum (FCS) in80 cm² flasks are transfected for four hours using lipofectamine(Invitrogen Life Technologies) according to the manufacturer'sinstructions (140 microliters Lipofectamine per 10 micrograms of DNA perflask) in serum-free medium at 37° C. After this incubation, cells arewashed, resuspended in rich culture medium, and the cells grown for fivedays. The supernatant is harvested for analysis of the secreted scFv-Fcfusion protein. A sandwich ELISA is used to quantify the amount of IgGproduced, using two antibodies directed to the Fc region. The scFv-Fcfusion proteins are purified using protein A affinity chromatographyusing a HighTrap column (Amersham Pharmacia) according to themanufacturer's instructions for IgG1), and the eluate concentrated viaMicrocon-YM30 concentrator (Amicon) and its buffer exchanged for PBS pH7.0. The occurrence of different scFv-Fc mixtures, six in total for thecells transfected with the three scFv-Fc genes, are furthercharacterized as described above in ELISA, and using viral isolates thatare specifically recognized by the antibodies, including European batvirus 2 for antibody JB and Lagos bat virus and Mokoa virus for antibodyJA, and strains CVS-11, CVS-24, PM, SHBRV and COSRV (Champion et al., J.Immunol. Methods (2000) 235:81-90). The presence of the M57 and JBbinding sites is confirmed using an anti-Id antibody (see also Examples14 and 22). Following this, the viral neutralization activity of themixture of three monospecific and three bispecific molecules (withoutpurification) is assayed for the presence of rabies virus-neutralizingantibodies using the rapid fluorescent focus inhibition test (RFFIT) asdescribed by Hooper et al., ASM Pres, WA, p. 1997. Essentially, serialdilutions are made of the supernatant containing the antibody mixture in96-well plates (Nunc), and a rabies virus dilution known to cause 70-80%infection of indicator cells added to each well. Controls are positiverabies-immune serum control samples and negative medium are alsoincluded. After one hour, to each well, 50,000 baby hamster kidney (BHK)cells are added and the culture incubated overnight at 37° C. Plates arethen washed once with ice-cold PBS and the cells fixed with ice-cold 90%acetone for 20 minutes at −20° C. Acetone is removed and to theair-dried plates 50 microliters of FITC-labeled anti-rabiesnucleoprotein monoclonal antibody (ab 1002 from abcam site or antibodyfrom Centocor, Malvern) is added. After one hour incubation at 37° C.,the plates are washed three times with water and analyzed under afluorescence microscope. The activity of each of the scFv-components isstudied by testing in this assay the neutralization of a variety ofdifferent rabies isolates, including the ones mentioned in Example 1.

The same plasmids, pscFv-Fc-JA, pscFv-Fc-JB and pscFv-Fc-M57, are alsosuitable for making stable transfectants. By selection using theneo-resistance gene and culturing and screening methods known to thosein the art, stable PER.C6™ derived cell lines expressing antibodies areobtained. Essentially 5×10⁶ PER.C6™ cells are transfected usingLipofectamine according to the manufacturer's instructions, and 3micrograms of DNA per plasmid. Cells are transfected with the 3micrograms of each plasmid separately, or with 1.5 micrograms each ofpscFv-Fc-JA and pscFv-Fc-JB, or with 1.5 micrograms each of pscFv-Fc-JBand pscFv-Fc-M57, or with 1 microgram of each of pscFv-Fc-JA,pscFv-Fc-JB and pscFv-Fc-M57, or with a control LacZ vector. After fivehours, the cells are washed and the medium is exchanged withnon-selective medium. The next day the medium is replaced with freshmedium containing 500 micrograms/ml G418 (Sigma-Aldrich) and also everynext two to three days, the culture medium is refreshed until clonesappear (15 to 20 days after seeding). Clones are picked and cloned outto limiting dilution conditions, such that two to three weeks later,clonal cell lines start appearing. These are expanded to larger wellsand flasks, and eventually the selective medium is omitted. Thesupernatant of these cell lines is harvested for analysis of thesecreted scFv-Fc fusion protein. As before, a sandwich ELISA (asdescribed in WO 00/63403) is used to quantify the amount of IgGproduced, using two antibodies directed to the Fc region. The scFv-Fcfusion proteins are purified using protein A affinity chromatographyusing a HighTrap column (Amersham Pharmacia) according to themanufacturer's instructions for IgG1. Purified scFv-Ig from variousclones is isolated, purified and tested in a series of assays. The firstis to analyze the presence of the two or three different scFv genes ofthe cell lines created, by amplifying the genomic DNA of these celllines with antibody JA/JB or M57 scFv orV-gene-specificoligonucleotides, and confirming the presence bysequencing the amplified material. The copy number of each of theintegrated antibody constructs is determined with methods such asSouthern blot or Fluorescent In Situ Hybridization (FISH). Secondly, themixture is biochemically characterized using SDS-PAGE and iso-electricfocusing. Alternatively, anti-idiotype antibodies or peptide mimitopesare used to delineate the compositions (see Example 12). The stabilityof the expression level, of the ratios between the different scFvcomponents, and of the composition of the antibody mixture produced bycell lines which produce the mix of three or six proteins is followedover time by these assays. Finally, binding and neutralization assaysare carried out, including antigen binding in ELISA and in fluorescencemicroscopy with infected cells and tissues, and in the RFFIT virusneutralization assay as described above. The biological activity of themixture is tested against a range of rabies isolates and the activitydetermined according to the international Units of Rabies Antibodies andreferenced to WHO reference Rabies Immunoglobulin (WHO Technical SeriesReport (1994) vol 848, p. 8; and vol. 840). By testing the biologicalactivity (virus neutralization) of a series of cell lines producingvariable quantities of the three scFv-Fc fusions, the most optimalmixture is identified. The mixtures are compared to the activity ofImmoGam® Rabies, the human immunoglobulin preparation used for passiveimmunotherapy (see alsohttp://www.aventispasteur.com/usa/product/pdffiles/!LE3439I.PDF). Theeffect of the bispecific component is tested by comparing theneutralization efficacy of the scFv-Fc protein mixture with the activityof comparable quantities of the (1) individual whole recombinantantibodies JA (IgG1), JB (IgG1) and M57 (IgG1), (2) mixtures of two orthree of these antibodies. Due to the discrepancy observed sometimesbetween in vitro and in vivo neutralization data, besides in vitroneutralization tests, it may sometimes be necessary to carry out in vivoneutralization tests using mouse protection experiments as described inDietzschold et al. (1992) PNAS 89:7252.

Example 4 Selection of Optimally Paired Variable Regions for TwoAntibody Variable Region Pairs by Optimizing the Light Chain VariableRegion

Antibodies M57 and JB are used in this experiment. Both have a lambdalight chain, of class I for JB and class II for M57, with homologybetween the two chains (FIG. 16). The antibody heavy chain variableregion genes of these two antibodies are cloned into vectorpFab-display, which resembles functionally pCES1 (H. J. de Haard et al.(1999) J. Biol. Chem. 274:18218-18230), and is a Fab fragment displayand expression vector. In this vector system, the variable heavy chainregion genes are cloned as VH-gene fragments; the vector supplies allFabs with a human gamma-1 CH1 gene. The Fd fragment is fused to two tagsfor purification and detection: a histidine tail for Immobilized MetalAffinity Chromatography (IMAC) and a c-myc-derived tag, followed by anamber stop codon and the minor coat protein III of filamentous phage fd.The antibody light chain is cloned as full VLCL fragment, for directedsecretion and assembly with the VHCH1 on the phage particle. Restrictionenzyme sites and the sequence of the polylinker region is indicated inFIG. 17. The cloning of the variable regions is carried out similarly asdescribed in Example 2, with oligonucleotides to amplify the VH regionand that append appropriate restriction enzyme sites. The resultingplasmids are designated pVH-M57 and pVH-JB, respectively.

These plasmids are used as recipients for a collection of human lambdachains derived from human donors. B lymphocytes are isolated from 2-L ofblood on a Ficoll-Pacque gradient. For RNA isolation, the cell pellet isimmediately dissolved in 50 ml 8 M guanidinium thiocyanate/0.1 M2-mercaptoethanol. Chromosomal DNA is sheared to completion by passingthrough a narrow syringe (1.2/0.5 mm gauge), and insoluble debris isremoved by low speed centrifugation (15 minutes 2,934×g at roomtemperature). RNA is pelleted by centrifugation through a CsCl-blockgradient (12 ml supernatant on a layer of 3.5 ml 5.7 M CsC1/0.1 M EDTA;in total four tubes) during 20 hours at 125,000×g at 20° C. in anSW41-rotor (Beckman). RNA is stored at −20° C. in ethanol. Random primedcDNA is prepared with 250 μg PBL RNA. RNA is heat denatured for fiveminutes at 65° C. in the presence of 20 μg random primer (Promega),subsequently buffer and DTT are added according to the suppliersinstructions (Gibco-BRL), as well as 250 μM dNTP (Pharmacia), 800 URNAsin (40 U/μl; Promega) and 2,000 U MMLV-RT (200 U/μl; Gibco-BRL) in atotal volume of 500 μl. After two hours at 42° C., the incubation isstopped by a phenol/chloroform extraction; cDNA is precipitated anddissolved in 85 μl water. From this material, the variable region genepools from the light chain lambda family are amplified using 4Vλ-specific oligonucleotides that preferentially pair to the lambda Iand II families (HuVl1A/B/C-BACK and HuVl2-BACK as in Table below) andwith two primers based in the constant regions (HuCl2-FOR and HuCl7-FORas in Table 1 below, combined in each reaction), and with PCR in avolume of 50 μl, using AmpliTaq polymerase (Cetus) and 500 pM of eachprimer for 28 cycles (one minute at 94° C., one minute at 55° C. and twominutes at 72° C.). All products are purified from agarose gel with theQIAex-II extraction kit (Qiagen). As input for reamplification tointroduce restriction sites, 100 to 200 ng purified DNA-fragment is usedas template in a 100 μl reaction volume, using the oligonucleotidesappropriately extended to provide the sites for cloning, ApaLI and AscI(last six primers of following Table). This amplified material ispurified, digested with AscI and ApaLI and two samples cloned into thetwo different plasmids pVH-M57 and pVH-JB.

HuV11A-BACK 5′-CAG TCT GTG CTG ACT CAG CCA CC- 3′ (SEQ ID NO: 25)HuV11B-BACK 5′-CAG TCT GTG YTG ACG CAG CCG CC- 3′ (SEQ ID NO: 26)HuV11C-BACK 5′-CAG TCT GTC GTG ACG CAG CCG CC- 3′ (SEQ ID NO: 27)HuV12-BACK 5′-CAR TCT GCC CTG ACT CAG CCT-3′ (SEQ ID NO: 28) HuC12-FOR5′-TGA ACA TTC TGT AGG GGC CAC TG- 3′ (SEQ ID NO: 29) HuC17-FOR 5′-AGAGCA TTC TGC AGG GGC CAC TG- 3′ (SEQ ID NO: 30) HuV11A-BACK-APA 5′-ACCGCC TC ACC AGT GCA CAG TCT GTG CTG ACT CAG CCA CC-3′ (SEQ ID NO: 31)HUV11B-BACK-APA 5′-ACC GCC TCC ACC AGT GCA CAG TCT GTG YTG ACG CAG CCGCC-3′ (SEQ ID NO: 32) HuV11C-BACK-A.PA 5′-ACC GCC TCC ACC AGT GCA CAGTCT GTC GTG ACG GAG CCG CC-3′ (SEQ ID NO: 33) HUV12-BACK-APA 5′-ACC GCCTCC ACC AGT GCA CAR TCT GCG CTG ACT CAG CCT-3′ (SEQ ID NO: 34)HuC12-FOR.ASC 5′-ACC GCC TCC ACC GGG CGC GCC TTA TTA TGA ACA TTC TGT AGGGGC CAC TG (SEQ ID NO: 35) HuC17-FOR-ASC 5-ACC GCC TCC ACC GGG CGC GCCTTA TTA AGA GCA TTC TGC AGG GGC CAC TG (SEQ ID NO: 36)

This cloning results in two libraries designated as Fab-VH-M57-VLn andFab-VH-JB-VLn.

Phage particles are made from cultures of these two libraries. Therescue of phagemid particles with helper phage M13-KO7 is performedaccording to (Marks et al. (1991), J. Mol. Biol. 222:581-597) on a 1-Lscale, using representative numbers of bacteria from the library forinoculation, to ensure the presence of at least ten bacteria from eachclone in the start inoculum. For selections, 10¹³ cfus (colony formingunits) are used with 10 micrograms/ml Rabies glycoprotein coated inimmunotubes (Maxisorp tubes, Nunc) or with 250 nM soluble biotinylated Gprotein. Antigen is biotinylated at a ratio of one to five moleculesNHS-Biotin (Pierce) per molecule antigen according to the supplier'srecommendations. Three rounds of selection are carried out with theselibraries. Detailed protocols for culturing and selecting phage displaylibraries have been described elsewhere (as in Marks et al. (1991), J.Mol. Biol. 222:581-597) and are well known to those working in the art.Briefly, the selection with the biotinylated antigen is carried out asfollows. Phage particles are incubated on a rotator wheel for one hourin 2% M-PBST (PBS supplied with 2% skimmed milk powder and 0.1%Tween-20). Meanwhile, 100 μl Streptavidin-conjugated paramagnetic beads(Dynal, Oslo, Norway) are incubated on a rotator wheel for two hours in2% M-PBST. Biotinylated antigen is added to the pre-incubated phage andincubated on a rotator wheel for 30 minutes. Next, beads are added andthe mixture is left on the rotator wheel for 15 minutes. After 14 washeswith 2% M-PBST and one wash with PBS, phage particles are eluted with950 μl 0.1 M triethylamine for five minutes. The eluate is immediatelyneutralized by the addition of 0.5 ml Tris-HCl (pH 7.5) and is used forinfection of long-phase E. coli TG1 cells. The TG1 cells are infectedfor 30 minutes at 37° C. and are plated on 2×TY (16 g Bacto-trypton, 10g Yeast-extract and 5 g NaCl per liter) agar plates, containing 2%glucose and 100 μg/ml ampicillin. After overnight incubation at 30° C.,the colonies are scraped from the plates and used for phage rescue asdescribed (Marks et al. (1991), J. Mol. Biol. 222:581-597). Culturesupernatants of individual selected clones harboring either rescuedphage or soluble Fab fragments are tested in ELISA with directly coatedantigen or indirectly captured biotinylated antigen via immobilizedbiotinylated BSA-streptavidin. Here described is the procedure withbiotinylated antigen for the detection of soluble Fab fragments. Forcapture of biotinylated Rabies glycoprotein, first biotinylated BSA iscoated at 2 μg/ml in PBS during one hour at 37° C. After three washeswith PBS-0.1% (v/v) Tween 20 (PBST), plates are incubated during onehour with streptavidin (10 μg/ml in PBS/0.5% gelatin) (24). Followingwashing as above, biotinylated antigen is added for an overnightincubation at 4° C. at a concentration of 3 μg/ml. The plates areblocked during 30 minutes at room temperature with 2% (w/v) semi-skimmedmilk powder (Marvel) in PBS. The culture supernatant is transferred tothese wells and diluted 1 or 5-fold in 2% (w/v) Marvel/PBS and incubatedfor two hours; bound Fab is detected with anti-myc antibody 9E10 (5μg/ml) recognizing the myc-peptide tag at the carboxyterminus of theheavy Fd chain, and rabbit anti-mouse-HRP conjugate (DAKO). Followingthe last incubation, staining ms performed with tetramethylbenzidine(TMB) and H₂O₂ as substrate and stopped by adding half a volume of 2 NH₂SO₄ the optical density is measured at 450 nm. Clones giving apositive signal in ELISA (over 2× the background), are further analyzedby BstNI-fingerprinting of the PCR products obtained by amplificationwith the oligonucleotides M13-reverse and geneIII-forward (as in Markset al. (1991), J Mol. Biol. 222:581-597).

Large-scale induction of soluble Fab fragments from individual clones isperformed on a 50 ml scale in 2×TY containing 100 μg/ml ampicillin and2% glucose. After growth at 37° C. to an OD₆₀₀ of 0.9, the cells arepelleted (ten minutes at 2,934×g) and resuspended in 2×TY withampicillin and 1 mM IPTG. Bacteria are harvested after 3.5 hours growingat 30° C. by centrifugation (as before); periplasmic fractions areprepared by resuspending the cell pellet in 1 ml ice cold PBS. After 2to 16 hours rotating head-over-head at 4° C., the spheroplasts areremoved by two centrifugation steps: after spinning during ten minutesat 3,400×g, the supernatant is clarified by an additional centrifigationstep during ten minutes at 13,000×g in an Eppendorf centrifuge. Theperiplasmic fraction obtained is directly used for determination of theaffinity by surface plasmon resonance and of fine-specificity in westernblot or virus neutralization studies.

Using the cited ELISA test, panels of antigen reactive Fabs areidentified for both M57 and JB. The Fabs are purified and their relativeaffinity for the antigen compared to the native antibody as Fabdetermined. All clones that are in a ten-fold reach of the affinity aresequenced. For sequencing, plasmid DNA is prepared from 50 ml culturesgrown at 30° C. in medium, containing 100 μg/ml ampicillin and 2%glucose, using the QIAGEN midi-kit (Qiagen). Sequencing is performedwith the thermocycling kit (Amersham) with CY5-labeled primers CH1FOR(5′-GTC CTT GAC CAG GCA GCC CAG GGC-3′ (SEQ ID NO:37)) and M13REV(5′-CAG GAA ACA GCT ATG AC-3′ (SEQ ID NO:38)). The analysis is done asdescribed above: the amino acid sequences of the two antibody VL sets,for M57 and JB, are compared to one another. Many of the selectedvariants are derived from the lambda 1 and lambda 2 family but carrysomatic mutations throughout the sequence. In each collection, a set of10 VLs are selected that are putative “common” candidates for pairing toboth VHs, and these are cloned via the common restriction sites ApaLIand AscI into the plasmid carrying the other VH. Thus, the VLCL of acandidate clone of library Fab-VH-M57-VLn is isolated usinggel-electrophoresis of the ApaLI-AscI digest and cloned into pVH-JB.This is carried out for all candidate VLs; the new combinations are alltested as before in ELISA for their pairing compatibility with thenon-cognate VH. The clone with highest affinity in both antibodies isdesignated VL-M57=JB. This procedure leads to the identification of alambda variable region light chain that in the Fab format can optimallypair with both the VH of JB and of M57.

Example 5 Selection of Optimally Paired Variable Regions for TwoAntibody Variable Region Pairs by Optimizing the Heavy Chain VariableRegion

For occasions where the two light chains of two given antibodies arevery different from one another, as is the case between antibodies ofkappa and lambda families, it is also possible to follow an alternativestrategy than the one described in Example 4. Herein we describe theselection of an optimally paired VL that will be pairing in a compatiblefashion with two VH variable regions. In the experiment, the major loopin the VH, the CDR3 that is both responsible for antigen binding andcontributes to the interaction with the light chain, is diversified.Other schemes can be followed, in which other VH residues known to bestructurally positioned at the VH-VL interface are mutated (exemplifiedin FIG. 18). This procedure may also be applied to multiple variableregion genes, using a chosen, preferably germ line encoded variableregion gene and multiple partner variable regions which are thenmutagenized and selected as in the following description.

The aim of the experiment is to find a JA-variant that will haveoptimally pairing behavior to VL-M57=JB. The JA antibody carries a kappachain instead of a lambda (FIG. 16), and replacement of its cognatelight chain with VL-M57-JB leads to a substantial loss of affinity.Therefore, it is the VH of this antibody that will be mutated, tocompensate for loss of affinity with the antigen, and to provide also aalso new potential interactions with the new VL. First, the VL-M57=JB iscloned as VLCL ApaLI-AscI fragment into pFab-display as described inExample 4; this yields plasmid pVL-M57=JB. The heavy chain of antibodyJA is amplified from pVH-JA (Example 2) using two primers: 5′-GTC CTCGCA ACT GCG GCC CAG CCG GCC ATG GCA GAG GTG CAG CTG TTG GAG TCT GGGGG-3′ (SEQ ID NO:39), and the reverse complement of the followingsequence, which is a mutagenic oligonucleotide that is spiked withmutations in the two residues preceding the CDR3 and throughout the CDR3region (in the underlined region; see also FIG. 18): 5′-C ACG GCC GTATAT TAC TGT GCG AAA GAT CGA GAG GTT ACT ATG ATA GTT GTA CTT AAT GGA GGCTTT GAC TAC TGG GGC CAG GGA ACC CGGG TCA CCG TCT CCT-3′ (SEQ ID NO:40).The spiking is carried out by the inclusion during the oligonucleotidesynthesis at the underlined residues, of mixes of 90% of the naturalresidue, and 10% of a mix with equimolar ratios of the four residues.The PCR is carried out as in Example 1 to yield a 350-400 by fragment,which is gel-purified, digested with SfiI and BstEII and cloned intopVL-M57=JB, to form a library of variants of JA, designatedFab-JA-YHmut.

This library is now rescued using helper phage and selections andscreenings are carried out on Rabies glycoprotein according to themethods described in Example 4. The resulting Fab clones that maintainantigen binding contain a VH-JA variant that is pairing-compatible withVL-M57=JB. Candidate Fabs are produced and purified, and their affinitydetermined as described in Example 4. The variable heavy chain mutant ofthe highest affinity is designated VH-JA*.

Example 6 Isolation of Antibodies Against Rabies Glycoprotein from aRandom Combinatorial Phage Library and Screening for Compatible VLBetween Binding Clones

Phage display libraries are a suitable source of antibodies for thepresent invention. Libraries that are suitable for the assembly of thepanels of antibodies include non-immune libraries (H. J. de Haard et al.(1999) J. Biol. Chem. 274:18218-18230), semi-synthetic libraries (deKruif et al. (1995) J. Mol. Biol. 248:97, and Griffiths et al. (1994)EMBO J. 13:3245-3260) and also immune libraries, which often display alower level of variable chain diversity. The first application presentedis to select antibodies to one antigen only, providing a mixture ofantibodies directed to the same antigen that can then be screened forpairing-compatible variable regions, and used to produce an antibodymixture. The second application concerns the selection of antibodies totwo different antigens. Methods to carry out selections and screeningsare well known in the art and are also described in Examples 4 and 5.Using selection on antigens, panels of antibody fragments specific for agiven set of antigens are obtained. For each of the panels the sequenceof VH and VL is determined. Thus, each antigen will have a set ofreactive antibodies. It is then possible to identify by visualinspection in each of the panels those antibodies that share a given VLor have highly related VLs between the different sets. The casesdescribed in Example 4 are also applicable here. In the best case eachset has at least one antibody with an identical VL as at least one otherantibody in the other sets. When this is not the case, a suitable VLthat matches a given VH is found by the methods described in Example 4:the VH is paired with a repertoire of VLs, of which the composition isdriven by the homology with a given VL or VLs. Alternatively, one VL ischosen and the non-matching VH is mutagenized as described in Example 5,to yield compatible pairs for all sets. The sequences are furtherinspected to find pairing-compatible variable regions that do not havesequence identity or homology. Variable heavy chains that pair withmultiple variable light chains and vice versa are identified. Such“promiscuous” pairings imply that the variable region involved binds tothe same antigen with any of several partner chains. To rapidly identifysuch variable regions, it is particularly useful to use semi-syntheticantibody libraries which have a limited number of positions which werediversified, as has been described for the human synthetic phageantibody library in Griffiths et al. (1994) EMBO J. 13:3245-3260.

In the first application, antibodies are selected against one antigen,the Rabies glycoprotein. The library described in Griffiths et al.(1994) EMBO J. 13:3245-3260, is selected on the Rabies glycoproteinantigen as described earlier. There are different sources of theantigen, including the material purified as in Dietzschold et al. (1996)Laboratory Techniques in Rabies, Eds Meslin, Kaplan and Korpowski, WorldHealth Organization, Geneva, p. 175. Alternatively, a source ofrecombinant Rabies Glycoprotein (G) of the appropriate type is used forthe coating. The sequence of rabies G is available to persons in the artand so are cloning, expression and purification techniques. A suitableformat is to use an immuno-adhesion-type of molecules, in which thesoluble part of the glycoprotein is genetically fused to animmunoglobulin Fc region, and the fusion protein expressed in eukaryoticcells (see also Chamow and Ashkenazi, Antibody Fusion Proteins, 1999,Wiley-Liss, NY). For phage selection, the immuno-adhesion isbiotinylated to be used in a selection as described in Example 4, orimmobilized by coating. Alternatively, selections are carried out onimmobilized (or biotinylated) Rabies virions, and selections are carriedout each round on virions derived from different Rabies strains, toobtain a panel of antibodies that recognize the most common epitopespresent in the different strains. These procedures yield a panel ofantibodies directed to the Rabies antigen, but the compatibility of thepairing of variable regions of the individual candidates has to betested.

We disclose here the use of the antibodies from the phage antibodylibrary described by Griffiths et al. (1994) EMBO J. 13:3245-3260, butfor the clones from other libraries the same principles apply. A panelof Fabs reactive with the Rabies glycoprotein is identified and theprocedure to find optimally pairing VH and VL combinations as describedabove carried out. As an alternative, independent of sequencing, toidentify optimally paired VH and VL pairs (that, for example, are missedin the sequencing analysis), the following empirical approach isfollowed. The variable light chains of a panel of 30 human antibodiesare shuffled, and the new combinations tested in a binding assay. Theshuffling is carried out by recloning the light chains present in theantigen reactive Fab clones which are based in the recombinedfd-DOG-2lox-plasmid, as ApaLI-AscI fragment into the same Fab-containingphage genomes cut with the same (unique) enzymes. This is an experimentthat is done in batch, with all 30 VL inserts and 30 VH-containingvectors mixed; sequencing is used to delineate the pairing of each VH-VLpair. ELISA is used to define which antibodies retain antigen bindingactivity and those clones are sequenced. The resulting combinationsprovide VH-VL which are pairing-compatible, the first class of which isformed by clones that share a VL or related VL; in that case one can bechosen plus the different VH genes for making Oligoclonics™ (see Example10). The second class contains clones with “promiscuous” pairing, andthe VH genes of these are combined with the VH and VL pairs of thoseFabs which are compatible with this tolerant VH.

The second application concerns the selection of phage antibodies on twodifferent antigens, as indicated in FIG. 2. The same procedures as werejust described for one antigen are followed, now to assemble two sets ofantibodies, one for each antigen. The same procedures are followed alsoto identify clones with an identical or similar variable regionsequence, or empirically, to demonstrate the existence ofpairing-compatible antibodies between the two sets of antibodies.

Example 7 Isolation of Antibodies Against Rabies Glycoprotein from aPhage Library with Limited Diversity and Screening Antibodies Which areNon-Competitive

Phage antibody scFv or Fab libraries that are formed by focusing thediversity in one variable region and keeping the other variable regioninvariable, preferably a germ line sequence, are particularly relevantto the invention. From such libraries it is feasible to isolateantibodies with a different heavy chain yet identical light chain, orvice versa (FIG. 3). Such antibodies are readily reformatted into anOligoclonics™ format according to the invention. In the art, it has beendescribed that antibodies that share the same VL gene but have differentVH genes and widely varying specificities can be obtained from phageantibody display libraries (Nissim et al. (1994), EMBO J. 13:692-698).

A sub-library of the semi-synthetic scFv library (de Kruif et al. (1995)J. Mol. Biol. 248:97) is used in the following example. This sub-librarycontains antibodies with diversity in the VH region only. Selections onantigen are carried out as described in the previous examples. UsingRabies glycoprotein as the antigen as described in Example 6, ten humanantibodies with different VH yet identical VL are identified. These areimmediately suitable for inclusion into Oligoclonics™ (Example 10). Insome instances it will be favorable to identify those antibodies thatrecognize different epitopes from the other antibodies in the mixture,and/or to obtain antibodies that recognize the same epitope recognizedby a given monoclonal and polyclonal antibody. The competitive nature ofthe selected ten scFv antibodies with the Rabies monoclonal antibody M57is determined in ELISA, using the set-up described in Example 2(essentially, with bound antigen, adding sample, and detecting using anHRP-labeled anti-c-myc antibody) in the presence or absence of the M57antibody. Competition experiments between the clones are readilyperformed using similar competition ELISAs with the phage-scFv particlesand the soluble scFv fragments. Besides this procedure to screen clonesfor a particular competition-behavior, it is also possible to influencethe selection outcome, either by using an antibody to block a site onthe antigen during the selection (preventing antibodies to or competingwith this epitope from being selected), or by using an antibody tocompetitively elute the fraction of phage antibodies that is bound tothe same epitope. Examples of both are known in the art and methods areapplicable here also to define suitable antibody combinations forinclusion in the Oligoclonics™ composition.

Example 8 Isolation of Single-Domain Antibodies Against RabiesGlycoprotein from a VL Phage Library, and Pairing with a SuitableVariable Region

Antibodies made in two steps are also suitable for the inclusion in theOligoclonics™ format and to make antibody mixtures. Rabies-specificsingle domain VL antibody fragments are selected from a phage displayedrepertoire isolated from human PBLs and diversified by DNA-shuffling, asdescribed in van den Beucken et al. (2001), J. Mol. Biol. 591-601(libraries B and C). Selection and screening experiments are done asdescribed in the previous examples. After the third round of selection,the pool of VLs is taken for combination with one VH segment (asdepicted in FIG. 4( e)). For this, the VL pool is recloned by PCR as anApaLI-XhoII fragment into pFab-display (FIG. 17) into which is cloned asingle human VH. The latter is a DP-47 germ line encoded variable regionwith short CDR3 sequence designated VH-N (SEQ ID NO:14), which isobtained by providing via PCR antibody clone FITC-B11 from Table IV inGriffiths et al. (1994) EMBO J. 13:3245.3260, with a short, five-residueCDR3 of amino acid sequence GGAVY (SEQ ID NO:41), and cloning this asSfiI-BstEII fragment into pFab-display. This CDR3 is found in manydifferent antibodies, and a short sequence with minimal length sidechains (except for the tyrosine) is chosen to minimize effects onantigen binding and pairing. The resulting mini-library is screened forthose antibody Fab fragments that maintain antigen binding. The threebest Rabies glycoprotein-specific VL genes are designated VL-G1, G2 andG3. Similarly, the principles of this approach are applicable tobuilding antigen-specific heavy chain fragments based on the VH domain,and providing these with a “neutral” VL, or even “neutral” partner VH.

Example 9 Selection of Antibodies with Pairing-Compatible VariableRegions by Intracellular Competition, and Expression of a Composition ofTwo or Three Fab Fragments with Pairing-Compatible Variable Regions

Selections with phage libraries are carried out using monoclonalantibodies as competitors during the formation of new phage particles.The selection biases the library selection towards variable region pairswith compatible pairing in the context of multiple variable regionsbeing expressed in the same host cell. The system relies on thesimultaneous expression of two or more Fab fragments, the variableregion of one of which is anchored onto a phage coat protein (FIG. 5).

First, the variable region genes of antibody M57 are cloned intopFab-Sol-pbr, a derivative of pFab-display (FIG. 17) with the samepolylinker, but no gIII, no M13 intergenic region and instead of pUC119the pBR322 backbone carrying the ampicillin resistance gene. Thevariable region genes of antibody JB are cloned in pFab-Sol-ACY-cat,similar in set-up as the previous one but carrying the Chloramphenicolresistance gene and based on the pACYC backbone. Both plasmids mediatethe expression of the soluble non-tagged Fab fragment under control ofthe lacZ promoter, and they are compatible with one another and can bemaintained in the same cell with antibiotic selection. Methods for thecloning have been described earlier; the sequences of these antibodiesare also included in the sequence listings below, thus it will bepossible for someone working in the art to clone these Fabs into thesepolylinkers such that upon induction with IPTG, both antibodies areexpressed in the periplasm of the culture. These two antibody Fabfragments form the competitors in this method. E. coli TG1 cellsharboring both plasmids are infected with phage harboring a library ofhuman Fab fragments, in which the heavy chain is anchored to the phagecoat and the light chain is provided as a soluble, non-anchored chain.The fd-based library from Griffiths et al. (1994) EMBO J. 13:3245-3260,which contains both VH and VL diversity is used for infection, theresulting bacteria start producing new phage particles and incorporatethe L and Fd chains expressed from this genome. Cells are grown to an ODof 1.0, the cells washed to remove produced phage, and the cellsincubated for four hours in 1 mM IPTG. During this time, competitionwill occur for pairing between the three variable heavy and lightchains, and there are many opportunities for mispairing. The phageproduced during this induction time will only recognize the nativeantigen, if the VH is tolerant to pair with any VL yet bind antigen, orwhen it exclusively pairs with the VL that is also encoded in thegenome. The phage is harvested, PEG precipitated, dissolved in PBS, andis now selected for binding to Rabies glycoprotein. Methods forselection have been described earlier. In both case the phage will beable to bind antigen, and be enriched in a selection round with antigen.The phage resulting from the selection is used to infect cells harboringthe two Fab-containing plasmids, and the cycle of induction, phagepreparation and selection is repeated. After five rounds of thisselection, the resulting Fab proteins are tested for antigen binding ina solid phage ELISA and recloned into the soluble expression vectorspFab-Sol-ACY-cata and pFab-Sol-pbr. E. colis are transfected with one ofthese plasmids and either the M57-containing vector or the JB-containingvector described above, or no additional vector. These cultures areinduced with IPTG (inducing expression of one or two Fab fragments), andthe resulting Fab fragments and Fab mixes analyzed for antigen bindingin ELISA. To confirm exclusive or tolerant pairing, the Fab fragmentsare purified using IMAC and tested in a capture assay with antigen asdescribed in Example 2. The selected variable region pair can be furtherused to build an Oligoclonics™ mixture with either M57 or JB variableregion genes (but not together), as in Example 10.

For making a mix of these three antibodies, the experiment is repeatedusing the VL-M57=JB from Example 4 instead of the two original lightchains VL-M57 and VL-JB. The result of the selection is a small numberof Rabies antigen-specific VH-VL pairs derived from the phage library.The best candidate according to affinity, with designated variableregions VH-PO1 and VL-PO 1, is further tested as above to confirm thatit is pairing-compatible with the VH-57, the VH-JB and the VL-M57=JB.Next, the following expression cassettes are introduced in the same E.coli host cell using the two plasmids described earlier for producingthe competing Fab, using cloning methods familiar to those working inthe art: in cassette (1), on one plasmid, the VL-M57=JB-CL and VH-CH1 ofM57; in cassette (2), the VL-M57=JB and VH-CH1 of JB (a second copy isprovided to obtain an excess of light chain for pairing with the twoheavy chains); and in cassette (3), on the other plasmid, the VL-PO1-CLand VH-PO1-CH1. Induction with IPTG leads to the production of a mixtureof Fab fragments with paired variable regions, which is then recoveredusing IMAC purification. Alternatively, protein G purification is used.Using the binding and other assays described in the earlier examples forRabies glycoprotein antibodies, the mixture is characterized. Thecontents of the mixture is dependent on the growth and inductionconditions of the bacteria and the primary amino acid sequences of theFab genes.

Example 10 Methods for Production of Oligoclonics™ in Eukaryotic Cells

A method for producing a mixture of antibodies in eukaryotic cellsaccording to the invention, using expression in a recombinant host cellof multiple VH and VL genes resulting in the production of VH and VLproteins capable of pairing to form functional bivalent and bispecificantibodies, named Oligoclonics™, is exemplified herein. The generalformat of a eukaryotic expression vector for human monoclonal antibodiesis shown in FIG. 19.

The VH and VL regions of human monoclonal antibodies specific for rabiesvirus obtained by any of the methods described in the previous examples,can be inserted into an eukaryotic expression vector containing theHATV20 leader sequence and all the coding sequences of the constantregions of human immunoglobulin heavy (for example, IgG1) and lightchains (for example, a kappa light chain) essentially as described (E.Boel et al. (2000), J. Immunol. Methods, 239:153-166). In this example,the following variable region genes optimized for pairing are used:VH-M57, VH-JB (non-modified variable region genes, from Example 2),VH-JA* (the optimized sequence of the VH of antibody JA, from Example5), and only one light chain, VL=M57=JB (from Example 4). The resultingplasmids encoding heavy and light chains are transfected into eukaryoticcells such as the human cell line PER.C6™ and in Chinese Hamster Ovary(CHO) to generate stable cell lines secreting antibodies. For this,published methods and methods known to persons skilled in the art areused (E. Boel et al. (2000), J. Immunol. Methods, 239:153-166 and WO00/63403). For the generation of stable PER.C6™ cells secretingantibodies, PER.C6™ cells are seeded in DMEM plus 10% FCS and in tissueculture dishes (10 cm in diameter) or T80 flasks with approximately2.5×10⁶ cell per dish or flask and kept overnight in an incubator at 37°C. and 10% CO₂. The next day, transfections are preformed in separatedishes at 37° C. using Lipofectamine (Invitrogen Life Technologies)according to standard protocols provided by the manufacturer. Theplasmids encoding the monoclonal antibodies can be mixed in variousratios and used at a concentration of 1-10 μg/ml. As controls, cells aresubjected to the transfection procedure in the absence of plasmids.

After four to five hours, cells are washed twice with DMEM and fed withfresh culture medium. The next day, the culture medium is removed andcells are fed with fresh medium containing 500 μg/ml of the antibioticG418. Cells are fed every two or three days with culture mediumcontaining 500 μg/ml of G418. After about 20 to 22 days after initiationof the experiment, a large number of colonies is visible and from eachtransfection, 300 clones are picked and grown individually in 96-wellplates and further expanded in 24-well, 6-well and T25 flasks. At thisstage, cells are frozen in liquid nitrogen and production levels ofrecombinant immunoglobulin are determined in an ELISA according tostandard procedures (e.g., E. Boel et al. (2000), J. Immunol. Methods,239:153-166 and WO 00/63403). At this stage of the culture procedure,G418 is no longer added to the culture medium.

To establish the presence of anti-rabies antibodies in a mixture, asolid phase anti-rabies ELISA is performed. For the rabies virus ELISA,rabies virus glycoprotein is purified according to standard procedures(Dietzschold et al., in F.-X. Meslin et al. eds., Laboratory techniquesin Rabies, World Health Organization, Geneva, page 175). Plates(polySorb™, Nunc) are coated with 5 μg/ml of glycoprotein diluted in PBSand 150 μl/well. The plates are then blocked with 5% powdered milk inPBS and washed in PBS containing 0.05% Tween20 (PBS-Tween) prior to theaddition of supernatant samples. Following incubation at roomtemperature for two hours, the plates are washed with PBS-Tween toremove unbound antibody present in the supernatant samples.Enzyme-conjugated or biotinylated secondary antibodies specific forvarious human heavy chain isotypes are added for one hour at roomtemperature and the plates are subsequently washed with PBS-Tween.Detection of secondary antibody is performed according to standardprocedures (e.g., J. M. Champion et al. (2000), J. Immunol. Methods235:81-90); see also previous examples. Other analysis methods aredescribed in Examples 3, 4 and 12.

Next, it is demonstrated that a clonal cell line accounts for theproduction of each of the binding specificities encoded by the plasmids,i.e., proving that a single cell is able to produce a mixture ofmultiple anti-rabies antibodies. For a limited number of colonies thatsecrete a mixture of all monoclonal antibodies, 30 clones selected fromthe initial panel of approximately 300, clonality is further establishedby subcloning under limiting dilution known to persons skilled in theart. Picked and expanded colonies are seeded in a 96-well plate at aconcentration of 0.3 cells/well in DMEM with 10% FCS and expanded.Colonies of cells are processed as described above and are referred toas subclones. Subclones are screened by PCR on genomic DNA for thepresence or absence of each of the three constructs. Furtherconfirmation of the presence of the constructs is obtained by nucleotidesequence analysis of the PCR products.

For a representative number of subclones, larger volumes are cultured topurify the recombinant human IgG1 fraction from the conditionedsupernatant using Protein A affinity chromatography according tostandard procedures. Purified human Ig from the various subclones issubsequently analyzed by SDS-PAGE, Iso-electric focusing (IEF) accordingto standard protocols (see, also, Examples 3 and 12).

Subclones that are shown to harbor the relevant plasmids are broughtinto culture for an extensive period of time to determine whether thepresence of the plasmids is stable and whether expression of theantibody mixture remains the same, not only in terms of expressionlevels, but, in particular, the ratio between the various antibodiesthat are secreted from the cell. Therefore, the subclone culture ismaintained for at least 25 population doubling times. At every four tosix population doublings, a specific production test is performed usingthe human Ig-specificELISA and larger volumes are cultured to obtain thecell pellet and the supernatant. The cell pellet is used to assess thepresence of the three constructs in the genomic DNA, either via PCR,Southern blot and/or FISH. The supernatant is used to purify therecombinant human Ig fraction as described. Purified human Ig obtainedat the various population doublings is subsequently analyzed asdescribed, i.e., by SDS-PAGE, Iso-electric focusing (IEF) and binding inthe inhibition ELISA.

Example 11 Method for Selecting Antigen-Specific Proteinaceous CompoundsUsing Mixtures of Encoding DNA

The basis for the mixtures of antibodies present in Oligoclonics™ areimmunoglobulin variable regions that encode human monoclonal antibodiesthat have been selected for their specificity, contain variable regiongenes with compatible pairing behavior and are thus compatible with theOligoclonics™ format. For example, antibodies that are encoded bydifferent VH genes and bind to different epitopes but share the same VLgene are suitable for the Oligoclonics™ format. Example 7 describes howsuch antibodies are obtained.

In this example, methods using eukaryotic expression systems to obtainhuman monoclonal antibodies with desired specificities and that sharethe same VL gene are described. Such “repertoires” of human VH genes arePCR-amplified from the B lymphocytes of human individuals and typicallyharbor 10⁸ to 10¹⁰ members. The method is known to persons skilled inthe art and has been described many times in the literature; theamplification of antibody genes is also exemplified for human V-lambdalibraries in Example 4. The source of B lymphocytes may be any lymphoidorgan including blood, bone marrow, tonsil, spleen, lymph node, etc. Theindividual may be pre-selected because it is expected that B lymphocytesproducing the antibodies of interest are enriched in those individualsbecause of, e.g., a prior infection with a micro-organism or because ofa prior immunization, or may be randomly picked. The VH genes may beused unaltered in their coding region or may be altered, particularly inthe CDR3 region to introduce novel specificities. Such VH genes areknown in the art as synthetic or semi-synthetic VH regions. Preferably,primers are used that selectively amplify members of a few VH genefamilies such as the large VH3 and VH4 gene families. Primers thatamplify members of more VH gene families may also be used in proceduresknown by persons skilled in the art. Amplified VH regions are clonedinto the eukaryotic expression vector for human monoclonal antibodies asdescribed in Example 10 and subsequently introduced into eukaryoticcells such as CHO cells or PER.C6 cells. The expression plasmid shown inExample 10 that harbors a VL gene is used (FIG. 7). There are twoalternatives: (1) the VL gene is co-transfected with the VH genes on aseparate plasmid or (2) an approach particularly suitable when only oneVL needs to be expressed the eukaryotic cells are already transfectedwith a human VL gene that is stably expressed. The eukaryotic cells aretransfected with the repertoire of human VH genes cloned into theeukaryotic expression vector for human antibodies. High plasmid DNAconcentrations are used to transfect the eukaryotic cells in order tointroduce multiple copies of VH genes into a single cell. As a result asingle cell will produce multiple antibodies, e.g., between 10 to 1000different human antibodies. In the first approach, transfections aretransient. In brief for PER.C6 cells, an 80 cm² tissue culture flaskwith cells is transfected by incubation for four hours with 140 μllipofectamine+10 to 1000 μg plasmid DNA in serum-free DMEM medium. Afterfour hours, the medium is replaced with DMEM+10% FCS, and the cells aregrown overnight at 37° C. Cells are then washed with PBS and the mediumis replaced with Excell 525 medium (JRH Bioscience). The cells areseeded at a concentration that results in the outgrowth of approximately100 transfected cells/well of a 96-well culture plate. After five to sixdays, the cell culture supernatant is harvested and assayed for thepresence of specific antibody by solid phase ELISA. The cells thatcorrespond to the supernatants that score positive in ELISA areharvested and the VH genes are amplified by PCR. Subsequently, theamplified VH genes are cloned into the eukaryotic expression vector forhuman monoclonal antibodies, described in Example 10. Thus, a restrictedrepertoire of human VH genes is obtained. In this example, 100 cellsthat each harbors 100 VH genes yield a maximum of 10⁴ VH genes. Thisrepertoire is transiently transfected into PER.C6 cells that harbor thesame VL gene using low plasmid DNA concentrations (0.1 to 1 μg/ml) suchthat on average a single cell harbors a single VH gene and transfectedcells are plated out under conditions such that only approximately tencells/well will grow out. After five to six days, supernatants arescreened in ELISA for specific antibodies and the cells corresponding topositive supernatants are harvested and used for PCR amplification ofthe VH genes. In this example, the maximum number of VH genes obtainedis approximately ten. Each VH gene is separately transfected into PER.C6cells and the VH gene encoding the desired antibody specificity isidentified by screening the supernatants of clones in ELISA.

In a second approach, the initial library of 10⁸ to 10¹⁰ VH genes clonedtogether with a single VL gene into the plasmid described in Example 10,is transfected into PER.C6 cells and plated out in T80 cell cultureflasks. After four to six days, the cells are harvested and mixed withred blood cells coated with the antigen of interest and individual cellsare monitored for the secretion of specific antibodies against thecoated antigen by the reverse hemolytic plaque assay, well-known in theart (e.g., F. Dammacco et al. (1984) Clin. Exp. Immunol. 57:743-51). Blymphocytes inducing plaques are visualized under a light microscope andpicked with a micromanipulator. The single transfected PER.C6 cell istransferred to an Eppendorf tube, lysed and subjected to single cell PCRto amplify the VH genes. The advantage of this approach is that only afew rounds of selection are needed to identify the VH gene of interest.

In a third approach, stable transfectants are used. After thetransfection as described above, large collections of clones are grownessentially as described in Example 10, with the exception that clonesare not plated out under limiting dilution conditions. Instead, thecells after transfection are plated in microtiter plates such that aftergrowth in the selective medium multiple clones per well arise (e.g., 100cell clones per well as indicated in FIG. 7). Each clone expressesmultiple species of heavy chains, and each well contains multipleclones. The supernatant of these cultures are tested for antigen bindingand the VH-genes are further enriched in cycles of PCR, cloning,transfection and screening, as described above.

The expression of multiple antibodies by a single transfected eukaryoticcell is improved in all of these approaches by introducinganti-repressor DNA elements in the plasmid constructs for the expressionof human monoclonal antibodies. Anti-repressor elements confer highlevel and stable expression of proteins in mammalian cells in a copynumber-dependent fashion (Kwaks et al. (2003), Nat. Biotechnol.21:553-558). The DNA fragments responsible for this effect are amplifiedfrom the clones described in this citation and introduced upstream ofthe heavy chain expression cassette. The human anti-repressor elementnr. 40 (SEQ ID NO:15) is amplified from the pSDH vector containing theelement (described in Kwaks et al.), using flanking oligonucleotidesthat also incorporate restriction sites suitable for cloning(5′-GTCCCTAGGAATTCGATCAAGAAA GCACTCCGGG-3′ (SEQ ID NO:42) and thereverse complement of 5′-CCTCATGATGTACATTAGAT CGAATTCGTAATACG-3′ (SEQ IDNO:43)). In this example, EcoRI (GAATTC (SEQ ID NO:44)) which is notpresent in this segment, is appended at both ends of the segment in aPCR reaction, and the fragment digested with EcoRI and cloned into anEcoRI-digested acceptor plasmid. In this example, the latter is achimeric plasmid of VHExpress and VLExpress, which is a composition madeby cloning the full VHExpress plasmid (FIG. 15), cut with KpnI andEcoRI, and inserting the VK expression cassette that was digested withthe same enzymes (described in Persic et al. (1997) 187:9-18). Theresulting plasmid, pABExpress40 contains both heavy and light chaincassettes with their respective transcriptional orientation in oppositedirections, and the anti-repressor element positioned in the middle ofthe two transcription units. A schematic map of the plasmid is shown inFIG. 22. This plasmid, pABExpress40 is used first in the cloning of theone chosen VL gene (using ApaLI and Pad cloning sites), resulting inpABExpress40-VL. This plasmid is used to receive the VH repertoiredescribed above (as BssHII-BstEII fragment) (all of these four sites areunique in pABExpress40 and pABExpress40-VL). The cloning of therepertoire is carried out as described for the lambda repertoire inExample 4, using in the PCR of IgM-primed cDNA a set of nineoligonucleotides labeled “VH-back” and the mix of four “VH-forward”oligonucleotides described in Table 1 of H. J. de Haard et al. (1999),J. Biol. Chem. 274:18218-18230. The material is re-amplified usingvariants of the nine oligonucleotides appended with 5′-TATC CGC GCG CACTCC-3′ (SEC) ID NO:45) and with the same VH forward mix, the productdigested with BssHII and BstEII and cloned into pABExpress40-VL. Thelibrary is subsequently used as described in the previous examples toisolate panels of antigen-binding clones. Similarly the vector is usedto construct the expression plasmid for given sets of antibodies, suchas the ones described in Example 10, further confirming that theflanking variable region genes by anti-repressor elements facilitatesthe efficient and stable production of multiple antibodies by a singlecell.

Example 12 Recovery and Analysis of Antibody Mixtures Using ELISAIncluding the Use of Anti-Idiotype and Peptide Mimotopes

Antibody mixtures containing Fc regions are recovered as indicated inExample 3 using Protein A affinity chromatography. Antibody fragmentswith Histidine tags are isolated using IMAC as described in Example 2.

The resulting protein mixtures are analyzed as follows. First instancewe consider the case of an antibody mixture composed of differentbinding sites directed to the same target antigen, with all antibodiesbeing the same isotype, carrying the same light chain, and the mixturecontaining both monovalent bispecific and bivalent monospecific IgG-typeantibodies. The following methods are available for analyzing themixture. The heavy chain variable region genes will yield differentamino acid compositions and allow the following non-antigen-dependentanalysis: (1) Isoelectric focusing gel analysis: this analysis relies ona different pI value for the various forms of the antibodies. In amixture of two IgGs and one bispecific, these three molecules will eachdisplay a unique isoelectric point. Proteins with a different pI areseparated via electrophoresis in a pH gradient. The method issemi-quantitative. If two proteins of the complex have only a minimaldifference in their pI value, it will be difficult to separate themusing this test, and the other tests cited are used. (2)Mass-Spectrometry analysis: this analysis relies on the differentialcomposition of the VH region, which, after digestion with proteolyticenzymes, yields a unique spectrum of peptides in MassSpec analysis. Thismethod is predominantly qualitative. (3) Binding analysis based onanti-idiotype antibodies or peptide mimics: this analysis requires theavailability of reagents that specifically recognize one antibodybinding site in the presence of the other binding sites in the mixture.Suitable for this analysis are anti-idiotype antibodies which uniquelyrecognize the idiotype of the antibody. In this example where theantibodies share a common light chain, the unique features of theidiotype are formed mainly by the heavy chain determinants.Anti-idiotype antibodies are selected using the individual monoclonalantibodies as antigen in a selection of a large phage displayed antibodylibrary using methods known to those in the art. Typically used are anon-immune antibody library (H. J. de Haard et al. (1999), J. Biol.Chem. 274:18218-18230), which yields Fab fragments, and a semi-syntheticphage antibody library (de Kruif et al. (1995) J. Mol. Biol. 248:97).Anti-idiotype antibodies are selected on immobilized M57 and JBantibodies from the cited non-immune antibody library. Using ELISAscreening of the selected phage antibodies on these two monoclonalantibodies used for the selection, anti-idiotype antibodies thatuniquely recognize one of the two binding sites are identified. Therespective Fab and scFv reagents selected from these library, areexpressed as antibody fragments and purified using standard methods, forexample, described in these citations and in Antibody Engineering(2001), Eds. Konterman and Dubel, Springer Lab Manual, and described inExample 2 for the scFv antibodies. The fragments are used in ELISA todetermine which idiotype is present in the mixture, which is carried outin a quantitative assay. The anti-idiotype antibodies specific for thebinding sites of M57 and JB are also used in virus competitionexperiments with the Oligoclonics™ preparation made in Example 10, todelineate the contribution of an individual binding site to thebiological activity of the antibody mixture. Alternatively, themonoclonal antibodies are used to derive idiotype-associated peptides,linear or conformational peptides derived from the sequence of theantigen and still reactive with the antibody, for example, via PepScananalysis, as was demonstrated for the rabies virus ˜neutralizingantibody MAb 6-15C4 (van der Heijden et al. (1993), J. Gen. Virol.74:1539-45). An alternative is to isolate peptide mimotopes, withsequences unrelated to the original antigen yet specifically binding tothe variable regions of the antibody. Provided the reaction is specificfor a given antibody in the context of the other antibodies in themixture, such peptides are also suitable for a specific analysis of theantibody mixture. Peptides with such unique reactivity to a givenantibody are selected from phage display peptide libraries using methodsessentially similar to those for phage antibody libraries. The bindingtest with the anti-idiotype antibodies and peptide-mimotopes isqualitatively or quantitatively, and a large series of binding tests arefeasible, including ELISA, RIA, Flow cytometric analysis, BIAcore, etc.

We also disclose the analysis of an Oligoclonics™ mixture comprisingmultiple antibodies, in which each of the original antibodies binds to adifferent antigen. This resembles the situation in which the antibodiesrecognize the same antigen or target, and anti-idiotype reagents orpeptide mimics are available. The analysis of multiple specificities ina mixture is carried out as follows, keeping in mind that antigen issynonymous for anti-idiotype. The reactivity to individual antigens istested in ELISA on all antigens separately, with standardized assaysusing the monoclonal antibodies and quantitative IgG ELISA test. Antigenis coated directly or indirectly, the plates incubated with the antibodymixture, and bound antibody detected with an anti-IgG reagent. Thisleads to a “specific” activity of the preparation, that is a reactivityin relative units of activity per antibody quantity. The amount ofbispecific antibody in the mixture is determined using a sandwich assaywith one antigen coated and a second antigen, preferably labeled withHRP, Alkaline Phosphatase or biotin, or detectable using anotherantibody specific for this antigen, provided to the plate after theantibody mixture was incubated with the first antigen.

If the antibodies present in the Oligoclonics™ mixture are bindingdifferent targets or different epitopes on the same target such thatthey are non-competitive, this feature can be used in an inhibitionELISA to determine the presence of the different antibodies in themixtures produced by the transfected clonal cell lines. Consider anOligoclonics™ made according to the methods of the previous examplesusing the antibodies specific for the Rabies glycoprotein isolated inExample 7 (which are non-competitive). For the inhibition ELISA, thesame procedures as described for the standard anti-rabies ELISA asdescribed above is used with some modifications. The Oligoclonics™mixture produced by a clonal cell line is characterized as follows.Before addition to the wells coated with rabies glycoprotein, thesupernatants of the transfected clonal cell line is diluted with anequal volume of a biotinylated rabies monoclonal antibody used to makethe mixture. The biotinylated rabies monoclonal antibody is added invarious concentrations, ranging from 0.1 to 10 μg/ml. Binding of thebiotinylated monoclonal antibody to the coated rabies glycoprotein isinhibited when the same non-biotinylated antibody is present in themixture produced by the clonal cell line. The binding of thebiotinylated antibody is visualized with streptavidin, conjugated to anenzyme. As a control for binding and degree of inhibition, variousconcentrations of the biotinylated monoclonal antibodies diluted with anequal volume of culture medium without the mixture of antibodies orusing the non-biotinylated antibody are used in the inhibition ELISA.This method is also suitable to screen the mixture of antibodies at avery early stage after transfection (as in Examples 10 and 11); thus,for each supernatant containing mixtures of antibodies, the presence ofindividual antibody specificities can be determined.

Example 13 Expression of Three Fab Fragments in the Same Eukaryotic Cell

For making a mix of these three antibodies, the expression experimentdescribed in Example 10 is repeated using the following antibody genes,of the M57, JB and PO1 antibody (the latter is formed by the VH-PO1 andVL-PO1 genes of Example 9). Anti-idiotype reagents are separatelyselected on M57 and JB whole antibodies using a non-immune antibodylibrary (see also Example 12). This yields anti-idiotype antibodies thatreact with either M57 or JB; these antibodies are also tested on the PO1to confirm specificity for either M57 or JB idiotypes. Similarly, thePO1 antibody is used in similar selections to obtain an anti-Id reagentfor the PO1 binding site. Next, the heavy chains of these threeantibodies, M57, JB and PO1, are cloned as VHCH1 fragments intoVHExpress while deleting the gamma-1 gene (thus encoding an Fd chainonly), yielding pEU-VH-M57, pEU-VH-JB and pEU-VH-PO1. The light chainsVL-M57=JB-CL and VL-PO1-CL are cloned into VKexpress (Persic et al.(1997) 187:9-18), while deleting the CK gene from the cassette. Firstthe light chain plasmids are introduced into PER.C6 cells and a clone isselected that stably produces over 2 micrograms/ml of both light chains(using methods described in Example 10). This cell line, designatedPL2-2, is subsequently transfected with the three heavy chain containingplasmids, and a large collection of cell lines is obtained that producea variety of levels of antibody Land Fd chains. The best candidatemixtures are purified on protein G affinity chromatography and testedfor binding and composition as described in the previous examples, andalso using the anti-Id reagents as described in Example 12. Theexperiments provide confirmation that multiple Fab fragments, withappropriately paired variable region genes, are expressed as highlyfunctional mixtures.

Example 14 Cloning and Expression of Three Antibodies Directed toDifferent Antigens as an Oligoclonics™ Mixture

Using the methods of the previous examples, antibodies with the samelight chain are isolated against three different antigen, TNF-alpha,Interleukin-1beta (IL-1beta) and Interleukin-6 (IL-6), using asemi-synthetic library scFv library from Example 7 and described in (deKruif et al. (1995) J. Mol. Biol. 248:97). In the selection,biotinylated recombinant cytokines (purchased from R&D Systems), areused, at decreasing concentrations during selection (250 nM, 100 nM and50 nM). From the panels of antibodies generated against each of thetargets after three rounds of selection, those scFv antibodies thatneutralize the activity of the cytokine are identified. For this, theantibody fragments are recloned into pSCFV and purified using IMAC as inExample 2. Biological assays used are well known to those skilled in theart and include a L929 neutralization assay for TNF-alpha. Neutralizingclones are identified against TNF-alpha, IL-1beta or IL-6. The potencyof neutralization can be improved by further affinity maturationtechniques. For example, the CDR1 and CDR2 of the VH can be mutagenizedand variants selected using phage display and tested for improvedneutralization activity. These three antibodies have an identical lightchain and have heavy chain variable regions that are distinct from oneanother, with most changes located in the CDR3.

The antibody variable regions are cloned into the eukaryotic expressiondescribed in Example 10, and essentially following the same procedure,CHO-cell lines identified that express mixture of the one light chainand three heavy chains. The analysis of the mixtures is carried outusing ELISA to demonstrate binding to three antigens in a subset of thecell lines identified. A clone stably producing all three antibodies inan approximate ratio of heavy chains of 2:1:1 is identified using thetechniques described in Examples 10 and 12. The cell lines are expandedand the mixture purified on Protein A and extensively tested todetermine its composition. Using ELISA tests in various formats, withindirectly coated biotinylated antigen, with directly coated antigen 1,adding sample, followed by biotinylated antigen 2 and detection withStrep-HRP, and using samples of the mixture that have been depleted onTNF, IL-1beta or IL-6-coated beads, is it shown that the mixturecontains three monospecific antibodies and three bispecific antibodies.The exact ratio between these six components is established by usingquantitative ELISA tests and by IEF analysis of the mixture, as shown inExample 12. The neutralization efficacy of the mixture for theindividual cytokines was confirmed with the assays as tested before. Theneutralization of these cytokines in more complex systems, for example,using mixed cell populations, may establish a synergistic effect of theneutralization of these components by the Oligoclonics™ mixture.

Example 15 In Vitro Pairing of Antibody Chains Produced in DifferentCells to Form Defined Antibody Mixtures

Alternatively, to the expression in one host cell, antibody mixture canalso be assembled ex vivo. The chains can be expressed separately andcombined with a set of potential partner variable regions for pairingand assembly of the molecule.

In this prophetic example, a mixture of Fab fragments withpairing-compatible variable regions will be made as follows. The heavychain variable regions of M57, JB and PO1 (Example 9) will first becloned separately into an appropriate pET expression plasmid, such thatthis will mediate the expression of the Fd chain tagged with sixHistidines inside the E. coli, as inclusion bodies. A suitable vectorcan be found in Novagen's pET Table, such as pET21d+ (see alsohttp://www.novagen.com/Includes/wrapper.asp?href=/SharedImages/Novagen/pETtable.htm&section=TechResources&subsectjon=TechLit&strsubsection=techresources).The cloning will then be carried out by PCR of the VHCH1-containingtemplates (from Example 9) using oligonucleotides to provide appropriatecloning sites and also the C-terminal Histidine tag. These threeplasmids will be introduced into separate E. coli host cells. Theexpression of the Fd fragments can then be induced and the proteindemonstrated to be present in inclusion bodies. The two light chainvariable regions, VL-M57=JB and YL-PO1 can also be suitably cloned intoa suitable pET vector (although, alternatively, they could be obtainedby secretion from a secretion vector like pFab-sol-pbr). Afterexpression of the separate light chains, they should also be retrievablefrom the intracellular fraction. To assemble the mixture of threefunctional Fab fragments, the following procedure can then be followed.First the approximate and relative quantities of the individual L or Fdchains is estimated by gel-electrophoresis and Western blot. Then five50-ml cultures of E. coli carrying one of five antibody variable regionsare grown and induced as described in the pET manual from Novagen. Afterinduction and growth, the pelleted cells of each of the chains can beresuspended in 8 ml 8 M urea (in PBS). After sonication, the fivesuspensions would be mixed in a ratio of approximately 1:1:1:4:2 forVH-M57, VH-JB, VH-PO1, VL-M57=JB, VL-PO1 (thus with a two-fold excess oflight chain over heavy chain, and more of the twice needed VL). Afterthis mixing of the denatured heavy and light chain variable regions, themix will be rotated head over head for two hours. Insoluble material maythen be removed by centrifugation for 30 minutes at 13,000×g. Thesupernatant is dialyzed against PBS with four buffer changes, andinsoluble protein further removed by centrifugation. The flow throughfraction, obtained by filtration through a 0.2 μm membrane, shouldcontain the refolded antibody mixture with pairing-optimized chains. Themixture may be further concentrated and purified, first using IMAC,which should retrieve all heavy chains and their paired light chains,followed by semi-preparative gel-filtration on a Superdex 75HR column(Pharmacia). The yield may be determined by measuring the opticaldensity at 280 nm (using a molar extinction coefficient of 13 for Fabs).The mixture may be further characterized by analyzing the binding to theRabies antigen. Since all functional Fabs should bind this antigen, astraightforward capture assay with antigen may be performed to determinethe level of functional binding sites. There are many alternativeprotocols to this procedure, including the use of other extractionmethods, other denaturation reagents, renaturation conditions andbuffers, etc. Alternatively, to this procedure, both chains may also besecreted, and re-assembled using the conditions described by Figini etal. (1994) J. Mol Biol. 239:68-78.

Example 16 Screening Antibody Mixtures Targeting Murine VascularEndothelial Growth Factor

The antibodies used in this example are described in WO 03102157A2(inventors Fuh and Sidhu). The antibodies were derived by in vitroselection of a display library in which only the heavy chain wasdiversified. The repertoire with a fixed light chain and variable heavychain was selected on murine vascular endothelial growth factor (mVEGF)and a large panel of antibodies binding mVEGF identified (Sidhu et al.,J. Mol. Biol. 2004, 338:299-310). The source of the antibody heavy andlight chain variable genes used in the repertoire was the humanizedantibody 4D5. Antibody 4D5 is a humanized antibody specific for acancer-associated antigen known as Her-2 (erbB2). The antibody includesvariable domains having consensus framework regions; a few positionswere reverted to mouse sequence during the process of increasingaffinity of the humanized antibody. The sequence and crystal structureof humanized antibody 4D5 have been described in U.S. Pat. No.6,054,297, Carter et al., PNAS 54:4285 (1992); the variable regionsequences of the heavy and light chains are also given in FIG. 14 andSEQ ID NO:23 of WO 03102157A2; finally the crystal structure of 4D5 isshown in J. Mol. Biol. 229:969 (1993) and online atwww.ncbi.nih.gov/structure, structure 1FVE.

An Oligoclonics™ mixture consisting of four different mVEGF-bindingantibody binding sites is obtained as follows. Antibodies with clonenumbers 4, 69, 73 and 74 as in Table 6, page 306 of Sidhu et al., J.Mol. Biol. 2004, 338:299-310, were selected on the basis of mVEGFbinding as scFv on phage and as Fab protein (same Table 6). Theantibodies share an identical light chain (of the Herceptin antibody,4D5; as described in WO 03102157A2), but have differences in their heavychain amino acid sequence as depicted in Table 6 of this paper.

The h4D5 antibody is a humanized antibody that specifically recognizes acancer-associated antigen known as HER-2 (ErbB2) developed as describedpreviously. The h4d5 VL gene is obtained by polymerase chain reactionusing the humAb4D5 version 8 (“hurnAMD5-8”; Carter et al. (1992) PNAS89:4285-4289) sequence and primers engineered to give rise to a 5′ ApaLIsite and a 3′ PacI site in the PCR product. The PCR product was cleavedwith ApaLI and PacI and ligated into the pABExpress vector (the vectordescribed in Example 11 and in FIG. 23 but without the STAR40 sequencecloned into the EcoRI site). This yields plasmid pAb-4D5-VL, whichencodes the expression of a functional 4D5 light chain (with humanCkappa constant region), and contains a polylinker region suitable forcloning VH regions. The VH regions from clones 4, 69, 73 and 74 are thencloned into this vector, using BssHII and BstEII restriction sites, andfollowing the cloning route described in the previous examples (byproviding the nucleotides encoding these restriction sites into the PCRprimers in such manner that the cloning will yield an in-frame insertionencoding a fully functional antibody variable domain). This yieldsplasmids pAb-IgG-04, pAb-IgG-69, pAb-IgG-73 and pAb-IgG-74.

These plasmids encoding heavy and light chains are transfected into thehuman cell line PER.C6™ to generate stable cell lines secreting multipleof the mVEGF-binding antibodies. For this, published methods and methodsknown to persons skilled in the art are used (E. Boel et al. (2000). J.Immunol. Methods, 239:153-166 and WO 00/63403). For the generation ofstable PER.C6™ cells secreting antibodies, PER.C6™ cells are seeded inDMEM plus 10% FCS and in tissue culture dishes (10 cm in diameter) orT80 flasks with approximately 2.5×10⁶ cell per dish or flask and keptovernight in an incubator at 37° C. and 10% CO₂. The next day,transfections are preformed in separate dishes at 37° C. usingLipofectamine (Invitrogen Life Technologies) according to standardprotocols provided by the manufacturer. The plasmids pAb-IgG-04,pAb-IgG-69, pAb-IgG-73 and pAb-IgG-74 are mixed in a 1:1:1:1 ratios andused at a concentration of 2.5 μg/ml each. As controls, cells aresubjected to the transfection procedure in the absence of plasmids, orwith just a single plasmid. After four to five hours, cells are washedtwice with DMEM and fed with fresh culture medium. The next day, theculture medium is removed and cells are fed with fresh medium containing500 μg/ml of the antibiotic G418. Cells are fed every two to three dayswith culture medium containing 500 μg/ml of G418. After about 20 to 22days after initiation of the experiment, a large number of colonies isvisible and from each transfection, 400 clones are picked and grownindividually in 96-well plates and further expanded in 24-well, 6-welland T25 flasks. At this stage, cells are frozen in liquid nitrogen andproduction levels of recombinant immunoglobulin are determined in anELISA according to standard procedures (e.g., E. Boel et al. (2000), J.Immunol. Methods, 239:153-166 and WO 00/63403). At this stage of theculture procedure, G418 is no longer added to the culture medium.

To establish the presence of at least one functional anti-mVEGF antibodyin a clone's culture supernatant, a solid phase ELISA is performed.Plates (PolySorb™, Nunc) are coated with 2.5 μg/ml of mVEGF (R&DSystems, recombinant Mouse VEGF120 and VEG164, both carrier free)diluted in PBS and 100 μl/well overnight at 4° C. The plates are thenblocked with 2% BSA in PBS for two hours and washed in PBS containing0.05% Tween20 (PBS-Tween) prior to the addition of cell supernatantsamples containing antibodies. Following incubation at room temperaturefor two hours, the plates are washed with PBS-Tween to remove unboundantibody present in the supernatant samples. Horseradishperoxidase-conjugated anti-human IgG is then added in PBS for one hourat room temperature and the plates are subsequently washed withPBS-Tween (2×) and PBS (2×). Detection of secondary antibody isperformed according to standard procedures and the absorbance determinedspectrophotometrically (see also previous examples). It is found that ofthe 400 clones screened, a substantial fraction produces a minimal IgGquantity.

Since only a limited number of colonies secrete a mixture of the fourmVEGF antibodies, 50 clones selected from the initial panel ofapproximately 400, that are strongly reactive in the IgG-ELISA,clonality is further established by subcloning under limiting dilution.Picked and expanded colonies are seeded in a 96-well plate at aconcentration of 0.3 cells/well in DMEM with 10% FCS and expanded.Colonies of cells are processed as described above and are referred toas subclones. While the initial transfection experiment used a ratio ofDNA for the four plasmids pAb-IgG-04, pAb-IgG-69, pAb-IgG-73 andpAb-IgG-74 of 1:1:1:1, the cell subclones still display a variety in theexpression levels for each of the antibodies. This is due to theirindependent expression regulation and their random integration into thegenome. Further, since the same selection marker is used on allplasmids, the subclones express at the most four antibody binding sites,but not necessarily all four of them. The precise number depends on thetransfection experiment; approximately 20-30% of the Ig-reactive clonesexpress multiple antibody heavy chains, and of these, approximately 20%express more than two antibody heavy chains. The methods to increasethese frequencies have been described earlier herein.

Screening to find the most optimal mixture of these four mVEGF-bindingantibodies, as Oligoclonics™ mixture with bivalent and bispecificcomponents, is done as follows. Optimal mixture here means with regardsto which of the four antibody binding sites are optimally present in themixture, and at which ratio they should be present. For the 50 subclonesas well as for one IgG-reactive clone from the control transfectantsmade with just one antibody encoding plasmid, larger volumes arecultured to purify the recombinant human IgG1 fraction from theconditioned supernatant. This is done using Protein A affinity columnchromatography according to standard procedures (Ed Harlow and DavidLane, Using Antibodies, A Laboratory Manual, 1999, ISBN: 0879695447).These mixtures and the monoclonal antibody controls are tested for theirneutralization activity on mVEGF in a ³H-thymidine incorporation assayusing human umbilical vein endothelial cells (Conn et al., 1990, Proc.Natl. Acad. Sci. U.S.A. 87:1323-1327). The inhibitory activity of eachof the mixtures is compared to the inhibitory capacity of the fourindividual monoclonal antibodies. Mixtures that display a higherinhibitory activity on a molar basis compared to the activity of themonoclonal antibody controls putatively contain multiple antibodies thatin combination mediate a synergic effect on the activity of VEGF. Next,assays that indicate the binding to mVEGF, the affinity of theinteraction of the mix, the competition in binding with receptor (Flt-1and KDR-1), are used. A binding assay is described above (solid phaseELISA). Assays to determine the relative affinity of the mixes aredescribed in Sidhu et al., J. Mol. Biol. 2004, 338:299-310, page 308(affinity measurements by competitive ELISA), with Fab andphage-displayed antibodies replaced with the mixtures of antibodies orthe monoclonal antibodies as controls. An increase in relative affinityindicates a strong synergistic activity between the antibodies in themixture, as described in Marks, Movelent Disorders, vol. 19, suppl. 8,2004, p. S101-S108, for antibody mixtures binding to nonoverlappingepitopes of neurotoxins. Other assays to demonstrate the activity of themixture of the antibodies on VEGF either in vivo or in vitro, are wellestablished in the field and are, for example, described in WO03102157A2, EP 0666868B1 and WO0044777A1.

Since VEGF displays activities in many processes, including mitogenesis,angiogenesis, endothelial cell survival, induction of metalloproteinasesand growth factors, regulation of permeability/flow, recruitment ofendothelial progenitor cells etc, any other single assays orcombinations of assays can be used to determine the effect of theantibody mixtures on the activity of VEGF. The antibody mixtures can bescreened in any of these assays, or combinations of assays, to findthose compositions that have an effect in a defined set of assays, orhave an effect in one but not in another assay. Further or instead ofthe in vitro assays, in vivo assays can be used to measure the overalleffect of the antibody mixture on the pharmacokinetics of the antigen,and demonstrate improved clearance as mechanism of the synergic activityof the multiple antibodies in the Oligoclonics™ mixture.

Mixtures are further characterized biochemically to find whichantibodies are present and in which ratio, as described in Example 12.

Example 17 Pairing-Compatible Antibodies for Producing a Mixture ofHER2/ErbB2-Targeting Molecules

Trastuzumab (Herceptin, or h4D5, or hu4D5, see Example 16) andpertuzumab (Omnitarg, humanized 2C4) are both recombinant monoclonalantibodies that target different extracellular regions of the HER-2tyrosine kinase receptor. Recently, it was shown that these antibodiessynergistically inhibit the survival of breast cancer cells in vitro(Nahta et al., Cancer Research 64:2343-2346, 2004). Herceptin is activeagainst HER-2 overexpressing metastatic breast cancers, leading to itsapproval in 1998 by the US FDA. In contrast to Herceptin, pertuzumabsterically blocks HER-2 dimerization with other HER receptors and blocksligand-activated signaling from HER-2/EGFR and HER-2/HER-3 heterodimers.On the other hand, trastuzumab blocks ErbB2 shedding while pertuzumabdoes not. Mixtures of antibodies directed to the same target antigen butthat display different or non-overlapping mechanisms of action will bevery valuable in the therapeutic arsenal, and production of suchmultiple antibodies in a commercial manner will become very important.In this example, we describe how pairing-compatible versions of thesetwo antibodies are isolated, and used to build an Oligoclonics™ with anexpected increase in potency and efficacy in tumor cell killing comparedto the original monoclonal antibodies.

Anti-HER2 antibodies 4D5 and 2C4 are described in WO 0100245A2 and inFendly et al., Cancer Research 50:1550-1558 (1990). The molecularstructure and sequence of the humanized version of antibody 2C4 isdescribed in Vajdos et al., J. Mol. Biol. 2002, 320, 415-428, in PDBdatabase reference 1L71, and in WO 0100245A2 (version 574 in Table 2 onpage 54, or rhuMAb2C4 in continuation of this document). For simplicityhere “2C4” is used to indicate the humanized version 574 of the murine2C4 antibody. Its structure, in complex with the first three domains ofErbB2, was recently published (Franklin et al., Cancer Cell, 5, 2004,317-328. The structure and sequence of h4D5 or Herceptin was describedby Cho et al., Nature 2003, 421, 756-760, and is deposited as 1N8Z inthe PDB database. Outside of the complementarity-determining regions(CDRs), pertuzumab is identical in sequence to trastuzumab (Carter etal., Proc. Natl. Acad. Sci. U.S.A. 89, 4285-4289, 1992); consequently,the local structure of the pertuzumab Fab in the ErbB2-pertuzumabcomplex is expected to be largely the same as that of the trastuzumabFab. To build a pairing-compatible single light chain that will restorea functional binding site when paired with the h4D5 VH but also whenpaired with the 2C4 VH, the following route is followed.

Designing Pairing-Compatible Light Chains

The amino acid differences between the light chains of hu4D5v8 (thehumanization variant described by Kelly et al., 1992, supra, indicatedby hu4D5 or h4D5 in the next section) and 2C4 have been mapped to be 11residues as highlighted in FIG. 23. In the CDR regions of the lightchains, there are four differences in CDR1, three in CDR2 and four inCDR3. In most straight forward to follow in the absence of structuraldata on the antibodies and their interaction with antigen, is to build alibrary of light chain that have been diversified at these positions,and screen or select for variant light chain that maintainantigen-binding behavior when paired with the heavy chains of bothantibodies, h4D5 and 2C4. The diversification can be chosen to containall possible 20 amino acids or a subset thereof, for example, allresidues but cysteine (which is not normally occurring at these 11positions), or a selected set of amino acids that frequently occurs inantibodies at these positions. The design of a light chain repertoirebased on all 11 amino acid differences between h4D5 and 2C4 is given inFIG. 23, in line HYB1.

A second approach to build a pairing-compatible variable hybrid lightchain region for two antibodies, is to further employ structuralinformation on the interaction of the antibodies with their respectiveantigen or antigens. In the example of h4D5 and 2C5, a wealth ofstructure-function information is available to guide the design of ahybrid light chain library. In this design, HYB2 in FIG. 23, all thelight chains in the designed repertoire retain all of the commonresidues between the two original light chains of hu4D5 and 2C4, and aselection of residues at the positions where the original two lightchains differ in composition, in which the selection is based onstructural information on the antibody-antigen interaction. While someof the design may be based on this information, it is also noted thatpoint mutations of h4D5 have been shown to dramatically effect thebiological behavior of the antibody. The antiproliferative activities ofthe humanized variants of 4D5, which differ only in maximally sevenamino acid residues, were found not to be strongly correlated withantigen affinity (Kelley et al., 1992, supra). Thus, it will be requiredto sample multiple versions of pairing-compatible light chains, and testthe biological activity of the combinations after the antigen-selectionand binding characterization to ensure maintenance of the biologicalactivity.

The following HYB2 library design was made, based on the followingobservations:

CDR1. The sequence plasticity of the antigen-binding site of Herceptinwas analyzed in a study by Gerstner et al. (J. Mol. Biol. 2002,321:851-862). From these studies it appears that for trastuzumabresidues N30 may be readily replaced by Serine (Table 1, Class 1mutation VL30, of Gerstner et al., supra). Serine is the residue used atthis position by 2C4. Thus, the pairing-compatible hybrid light chain isdesigned to contain Ser at position 30. The rest of the CDR1 is takenfrom the Herceptin light chain, as this region appears to be irrelevantfor antigen binding in 2C4 (Franklin et al., supra).

CDR2. By alanine-scanning and homolog-scanning of the Fab2C4 antibody itwas revealed that most of the side-chains that contribute to antigenbinding are located in the heavy chain (Vajdos et al., supra). This wasrecently confirmed by the crystal structure of the antibody in complexwith antigen: the light chain of pertuzumab Fab makes only a fewcontacts with ErbB2, mostly via CDR L2 (possibly via residue 55) andsome via L3 (Franklin et al., supra). Some of 2C4's residues in thisregion may be converted to h4D5's residues without loss of affinity, assuggested by experiments with humanized versions of 2C4 described in WO0100245A2 (page 54), in particular, what may be possible is to chooseh4D5's VL's residues at positions 54 and 56. The Phe at position 53 inHerceptin appears to be relatively conserved, with some presence of Trp,while the other positions in this CDR region were not tested. Since someof these CDR2-based residues may also be important for positioningneighboring heavy-chain-based residues for antigen binding, in thehybrid light chain design, the three residues which are differentbetween h4D5 and 2C4 are diversified fully, such that the selectionprocess can identify which of the 8000 combinations will yield apairing-compatible light chain.

CDR3. Tyrosine 91 of 2C4 is said to be important for antigen binding(Franklin et al., supra) but its substitution with phenylalanine (F) isacceptable (Vajdos et al., supra). Herceptin at this position in thelight chain besides its original residue histidine tolerates severalother aromatic side chains including Phe, Tyr and Trp (Table 1, page 854in Gerstner et al., supra). Thus, the hybrid light chain is designed tocontain Phe at position 91 (FIG. 23). For 2C4 antigen binding of theother residues of the H3 loop is relatively resistant to mutagenesis asin Gerstner et al., with the exception of the Pro at position 95. Butthis residue is shared between the Herceptin and 2C4 antibody lightchains. In the interaction of Herceptin with antigen there are morelikely interactions of the CDR3 regions with antigen, thus in the hybridlight chain, all but residue 91 is taken from Herceptin-VL (FIG. 23).

In the final HYB2 design, amino acids are taken for 6 out of 11positions from the h4D5 VL, 1 out of 11 from the 2C4 VL (pos. 30), oneis a residue not found in either VL (pos. 91) and the three are to berandomized (in CDR2).

HYB1 Library Construction and Selection of Pairing-Compatible VLs

The two libraries of light chains are constructed as follows. In theHYB1-designed VL library, 11 residues are randomized, implying that thetotal theoretical amino acid diversity (20exp11) is much larger than canbe readily screened. To sample the diversity in this library, a powerfulselection method is, therefore, used. The heavy chains (VH) of h4D5 and2C4 are cloned into the SfiI-BstEII cloning sites from pCES1 (de Haardet al., 1999, J. Biol. Chem. 274, 18218-30) using PCR andoligonucleotides binding to the 5′ and 3′ end of the nucleotidesequences of the VH genes and introducing SfiI and BstEII sites atappropriate sites for in-frame cloning (as described for antibody VHgenes in de Haard et al., supra; the BstEII site is already present inthe JH region of both h4D5-VH and 2C4-VH). The template for the PCR ofthe VH of h4D5 is plasmid pAK19 carrying the humanized 4D5 variantnumber 8, hu4D5-8, described in Kelly et al., 1992, Biochemistry31:5435-5441, Table 1. The nucleotide sequence of this clone isessentially described in Carter et al. 1992, P.N.A.S., 89:4285-4289, inFIG. 1, as huMAb4D5-5, with two alterations (V102Y in CDR3 of the VH,and E55Y in CDR2 of VL, as described in Kelly et al., 1992, supra). TheVH sequence can also be extracted as SfiI-BstEII fragment from SEQ IDNO:17 as described below. The template for the PCR reaction of the VH of2C4 is plasmid pC2C4, described on page 425 of Vajdos et al., supra. TheVH sequence can also be extracted from the NcoI-BstEII insertion insidethe larger BssHII-NotI-fragment from SEQ ID NO:17. The cloning of thePCR products into pCES1 is carried out as described for human antibodyheavy chain VH pools and using standard cloning procedures. pCES1 is aphagemid vector that is suitable for the expression of Fab fragments inE. coli and for the display of Fab fragments on the surface offilamentous phage (de Haard et al., 1999, supra). Two plasmids withcorrect insert are identified by sequencing the insertion and junctionregion and the resulting plasmids named pCES-VH-h4D5 and pCES-VH-2C4.These are the acceptor plasmids for the light chain repertoire, HYB1.The VLCL coding region of hu4D5v8 is amplified using specificoligonucleotides priming in its 5′ and 3′ region and introducing ApaLIand AscI restriction sites as described in de Haard et al., supra, forhuman VLCL chains. As template pAK19 carrying the humanized 4D5 variantnumber 8 (hu4D5-8, described in Kelly et al., 1992, Biochemistry31:5435-5441, Table 1) is used. The PCR product is cloned as ApaLI-AscIfragment in pCES-WI-h4D5, to yield pCES-Fab-h4D5. This encodes afunctional h4D5 Fab fragment. HYB1 is produced using described methodswith “stop” template versions of this plasmid. The stop template versionis made by replacing one codon in each of the CDR1, CDR2 and CDR3 of thehu4D5-v8-VL with TAA stop codons. Methods to diversify the VL-templatehave been extensively described in the literature including in WO03102157A2, in Directed Mutagenesis, a Practical Approach, Ed. M. J.McPerson, IRL Press 1991. The method used here is the Kunkel method;this yields the stop template of the VL in plasmid pCES-Fab-h4D5-3ST.The stop template version of h4D5-VL is used as a template for theKunkel mutagenesis method (Kunkel et al. 1987, Methods in Enzymol.154:367-382), using mutagenic oligonucleotides designed tosimultaneously repair the stop codons and introduce mutations at thedesigned sites. Mutations in all three CDRs of the VL are introducedsimultaneously in a single mutagenesis reaction. This is extensivelydescribed in Sidhu et al. 2000, Methods Enzymol. 328:333-363. Themutagenesis reaction is electroporated into E. coli SS320 (Sidhu et al.,supra), and the transformed cells are grown overnight in the presence ofM13-VCS helper phage to produce phage particles that encapsulated thephagemid DNA and displayed Fab fragments on their surfaces. Methods forphage-display library manipulation, selection and screening of cloneshave been described in the literature, for example, see de Haard et al.,supra; Vajdos et al., supra and also the other examples). The resulting4D5-HYB1 library contains greater than 1×10⁸ unique members. This4D5-HVB1 library is selected twice on HER2 antigen as described inVajdos et al., supra, to yield a population of more than 65% ofantibodies with antigen-binding activity. These antibodies share theirVH region, but most carry different light chains. The light chains ofthis population are obtained as ApaLI-AscI fragment (VLCL), and clonedas a pool into pCES-VH-2C4. This new library now contains a subset ofthe light chains of HYB1 that are likely to be compatible with antigenbinding in the context of h4D5. The library is selected once on antigen,and clones identified that mediate antigen binding. Light chains withidentical amino acid sequence and that mediate antigen-binding whenpaired with the h4D5-VH and with the 2C4-VH are identified by sequencinga panel of Ag-reactive clones from the selected h4D5-HYB1 library, andof Ag-reactive clones from the selected 2C4 sublibrary, and comparingthe sequences. Besides using antigen-reactivity in phage ELISA asreadout, the reactivity of the Fab fragments is tested in ELISA (asdescribed in de Haard et al., supra). This leads to the identificationof a panel of VLs that display are functionally pair with both VH-h4D5as well as VH-2C4. Within the panel the best VL is identified bydetermining the affinity of the interaction and the biological activityof the two respective Fab fragments. Methods for affinity determinationand biological activity of anti-HER2 Fabs are described in Kelley etal., 1992, supra, and Gerstner et al., 2002, supra, and are describedfurther below.

HYB2 Library Construction and Screening of Pairing-Compatible VLs

The HYB2-designed VL library contains 8000 variants only. Here adifferent route is followed to allow simultaneous expression, anddetection of antigen-binding variants, of h4D5 and 2C4 WI containingantibodies. First, the VL in pCES-Fab-h4D5 is mutated by Kunkelsite-directed mutagenesis (Kunkel et al., supra) with Asparagine 30changed to Serine (N30S), and Histidine 91 changed to Phenylalanine(H91F), according to the design depicted in FIG. 23. Of the resultingclone, p4D5-VLmut, phage and Fab are produced and tested for binding ina dilutions series for binding to Her-2 (extra-cellular domain) coatedplate phage ELISA, to confirm that h4D5 maintains a minimalantigen-reactivity. Next a stop-template version is made from thisplasmid, by replacing one codon in the CDR2 of the VL with a TAA stopcodon (residue 55, tyrosine is mutated from “tat” to “taa”; this residueis said to be required in order to attain the antigen affinity of thehumanized h4D5 antibody, Kelley et al., 1992, supra, thus will need tobe fixed to “Y” to restore the reading frame and antigen-binding), Thisstop template version of the light chain of h4D5v8 is cloned intopSCFV-3 (Example 2 and FIG. 14B), by amplification of the VLCL regionfrom the CDR2 stop-template. The design of the oligonucleotides used inthis amplification is such that the whole VLCL segment is amplified andthat after digestion the segment can be directionally cloned for inframe expression of the light chain under control of the arabinosepromoter of pSCFV-3, and without any C-terminal tags. Briefly the VLCLis amplified with primers binding to the 5′ and 3′ end of the cassetteand at the 5′ providing a long overhang in two PCR reactions to encode aregion of approximately 90 nucleotides encoding ribosome binding sites,start codon and bacterial leader sequence, to produce an EcoRV-EcoRIfragment that is cloned into the PacI-EcoRI sites bordering the thirdexpression cassette in pSCFV-3. This plasmid, pVLmutST, is used asacceptor for the two heavy chains, after an internal BstEII site atposition 143 of the insert was removed. The sequence of the finalPacI-EcoRI insert is given in SEQ ID NO:16. The heavy chains 2C4 andh4D5v8 are cloned in two steps as VH-CH1 fragments into pSCFV-3 (FIG.14B) to yield plasmid p2Fab-HER2 as indicated in FIG. 24. First the h4D5VHCH1 region is amplified from pCES-WI-h4D5 and cloned as SfiI-BssHIIfragment into pVLmutST. The design of the primers is such that they,after cloning, arrange appropriate reading frames with leaders and tagsin pSCFV3, to yield the final junctional sequences as depicted in SEQ IDNO:17. Secondly, the 2C4 heavy chain VHCH1 is amplified from pCES-VH-2C4and cloned as BssHII-NotI fragment into this plasmid. Similarly, thedesign of the primers is such that they, after cloning, arrangeappropriate reading frames with leaders and tags in pSCFV3, to yield thefinal junctional sequences as depicted in SEQ ID NO:17. This finalplasmid, p2Fab-Her2, provides the expression of both heavy chainvariable domains as Fd chains (linked to human gamma-1), and theexpression of a yet stop codon containing light chain. The sequence ofthe HindIII-NotI and PacI-EcoRI inserts of p2Fab-HER2 is given in SEQ IDNOS:17 and 16, respectively. The heavy chains of the two humanizedantibodies, h4D5-version 8 and 2C4-version 574 are provided as fusionsto the human CH1 domain, the Myc and VSV tag, respectively, and aHIS-tag for IMAC purification. The light chain in format VLCL isessentially derived from h4D5 but carries two designed VL mutations atpositions 30 and 91, a stop codon in the CDR2, and has an internalBstEII site removed without amino acid change.

Plasmid p2Fab-HER2 is used as a template for the Kunkel mutagenesismethod (Kunkel et al. 1987, Methods in Enzymol. 154:367-382), usingmutagenic oligonucleotides designed to simultaneously repair the stopcodon in the VL-CDR2 and introduce mutations at the three designed sitesin CDR2, as indicated in FIG. 23. After electroporation and plating (asbefore), a small library of 50,000 clones is screened forpairing-compatible VL versions as follows. In the plasmid p2Fab-HER2,all three variable region genes are linked to a unique epitope tag thatprovides a way for their specific detection. Individual clones arepicked into 96-well plates (Nunc) and induced to express both heavychains and the one light chain, using conditions as described in Example4, with the exception that arabinose is also added as inducer at thesame time as the IPTG. The next day the supernatant of the cultures istested for the presence of HER2 reactive Fabs, in an ELISA essentiallyas in Example 4. Multiple assays are carried out with the same sample,using either anti-myc or anti-VSV secondary reagents to detect thepresence of the h4D5-Fab or the 2C4-Fab, respectively.

A dual-reactive clone designated 3-8E3, which binds HER-2 in ELISA withboth the anti-VSV and anti-Myc tag reagents, is chosen for furtheranalysis. The Fab mixture of this clone is expressed to 10-L scale leveland purified from E. coli Supernatants according to Kelley et al., 1992,supra, page 5435-5436. Briefly, the culture supernatant is microfilteredby tangential flow filtration, concentrated by ultrafiltration andfiltered over DEAE-Sepharose-FF. The antibody mixture in theflow-through fraction is subjected to affinity chromatography onProtein-G-Sepharose-FF. The Fab mixture is eluted with 0.1 M glycine, pH3.0. The total protein concentration is determined by A₂₈₀ measurementsusing an ε₂₈₀ of 67 mM⁻¹ cm⁻¹.

The binding constant of individual Fabs or the apparent binding constantof the Fab mix are measured by ELISA essentially as described by Vajdoset al., 2002, supra, on page 426. Briefly, NUNC 96-well maxisorbimmunoplates are coated overnight at 4° C. with HER2-ECD (1 microgram/mlin 50 mM carbonate buffer, pH 9.6), and the plates blocked for one hourat room temperature with 0.5% BSA in PBS-0.05% Tween 20. Serialdilutions of Fab protein are incubated on the HER2-ECD coated plates fortwo hours at room temperature, and the plates washed. Bound Fab isdetected with biotinylated murine anti-human kappa chain antibodyfollowing by streptavidin—horseradish peroxidase conjugate (Sigma),using 3,3′,5,5′-tetramethyl benzidine (TMB) as substrate (Kirsgaard andPerry Laboratories, Gaithersburg, Md.). The actual binding constant ofone Fab in the mixture of two Fabs is measured by replacing thebiotinylated murine anti-human kappa chain antibody of the above testwith biotinylated anti-MYC-tag (for h4D5) or biotinylated anti-VSV tag(for 2C4) antibodies (antibodies similar to those described in Example2). Titration curves are fit with a four-parameter non-linear regressioncurve-fitting program (KaledaGraph, Synergy Software) to determine theEC50 values, the Fab concentrations corresponding to half-maximalbinding signals. Examples for h4D5, 2C4 and the 3-8E3 mixture is givenin FIG. 25. The 3-8E3 mix is confirmed to contain two functional Fabantibody fragments, h4D5* and 2C4*, in which the * indicates that thelight chain variable region is different from the two original humanizedlight chains of h4D5 and 2C4 (in FIG. 24), The ratio of the two Fabantibodies that are present in the 3-8E3 mix is analyzed byelectrospray-ionization mass spectrometry essentially as described inKelley et al., 1992, supra. There is a difference in the molecularweights of the Fabs on the basis of the heavy chains of 2C4 and h4D5differing in approximately 68 dalton, well above the standard deviationof the assay (in the range of three to seven dalton).

The biological activity of the Fab mixtures is compared with that of theindividual monoclonal Fab fragments. The growth inhibitorycharacteristics are evaluated using the breast cancer cell line, SK-BR-3(see Hudziak et al., 1989, Mol. Cell. Biol. 9:1165-1172), using theassay conditions described on page 50 of WO 0100245A2. An exemplarygraph in FIG. 25 shows the growth inhibition curves for h4D5 Fab andmixtures of 4D5* and 2C4* (see Example 17) that utilize differentpairing-compatible light chains, indicated with VL1 to VL7. The Fabs arefurther evaluated for their ability to inhibit HRG-stimulated tyrosinephosphorylation of proteins in the Mr 180,000 range from whole-celllysates of MCR7 cells, which are known to express all known ErbBreceptors (as in WO 0100245A2, page 50-51). As a control, 2C4 as Fab isused; this antibody is very effective in disrupting the formation of thehigh affinity HER2/HER3 binding site on MCF7 cells.

Once the activity of the Fabs in the mixture confirmed, the selected,pairing-compatible VL of 3-8E3, is used to build an Oligoclonics™ of theIgG format, essentially as described in the previous Example 10. Thisresults in the production of 30 cell clones each producing a mixture ofthe bivalent h4D5* and 2C4* antibodies, and the bispecific combination;the IgGs are purified from the cell supernatants by protein A columnchromatography as described above, and the concentration of the totalIgG present in the mixtures determined. The biological activity of theresulting IgG-mixtures is tested as in Nahta et al., Cancer Research64:2343-2346 (2004), using a growth inhibition assay of BT474 breastcancer cells as described on page 2343 of this paper. Briefly BT474cells are treated in triplicate with two-fold serial dilutions of theIgG mixtures in the range of 0.1 to 25 ng/ml. After five days, cells aretrypsinized and counted by trypan blue exclusion. The growth inhibitionis calculated as the fraction of viable cells compared with untreatedcultures. As controls, the original antibodies hu4D5-v8 (trastuzumab)and 2C4 (Pertuzumab) are used, as well as a 1:1 mixture of thesemonoclonal antibodies. The mixture with the most synergic activitybetween the two binding sites is identified based on the dose-effectplots as described in the legend of FIG. 1 on page 2344 in Nahta et al.,2004, supra. Other tests to confirm the synergistic activity aredescribed in this paper (in vitro tests: apoptosis induction, Aktsignaling), in WO 0100245A2 (in vitro tests and in vivo tests, such ashuman tumor xenograft models described in Examples 5 to 7 and in FIGS.10 to 13) and in Franklin et al., 2004, supra (in vitro HER2/HER3heterodimerization using COS7 transfected cells).

Other examples of antibodies that can be combined with one or both ofthese anti-ErbB2 antibodies are antibodies with pairing-compatiblechains that function as an anti-angiogenic agent (e.g., an anti-VEGFantibody); target the EGF-receptor (or ErbB1; e.g., C225 or ZD1839); orthat are anti-ErbB2 antibody that strongly induce apoptosis, such as 7C2or 7F3 (WO 0100245A2). Pairing-compatible light chains are identifiedusing the methods described in this invention.

Example 18 Pairing-Compatible Antibodies to Produce a Mixture ofHepatocyte Growth Factor/Scatter factor (HGF/SF)-Targeting Antibodiesthat Block Multiple Biological Activities

HGF/SF is a ligand that binds to the Met receptor tyrosine kinase.HGF/SF is composed of an α chain containing the N-terminal domain andfour kringle domains covalently di-sulfide linked to the β chain.Binding of HGF/SF to the Met receptor tyrosine kinase induces multiplebiological activities, including cell proliferation and cell invasion,and outgrowth of blood vessels (angiogenesis). In addition, binding ofHGF/SF to Met prevents programmed cell death (reviewed in C. Birchmeieret. al. Nat. Rev. Mol. Cell Biol. 4:915-925 (2004). The Met receptor isexpressed by many solid tumors and Met-HGF/SF signaling has been shownto be involved in tumor development, invasion and metastasis (J. M.Cherrington et al., Adv. Cancer. Res. 79:1-38 (2000); S. Rong et. al.,Mol. Cell Biol. 12, 5152-5158 (1992).

Monoclonal antibodies against HGF/SF have been produced to study theircapacity to block the diverse biological activities of HGF/SC (B. Cao etal., Proc. Natl. Acad. Sci. U.S.A., 98, 7443-7448 2001). The antibodieswere produced by immunizing mice with human HGF/SF and generatinghybridomas secreting monoclonal antibodies. The polyclonal serum frommice immunized with HGF/SC showed potent neutralizing activity of allbiologic activities of HGF/SF. In contrast a large panel of monoclonalantibodies that bind to the human HGF/SCF was shown to lack the capacityto completely block all biological activities of HGF/SC (B. Cao et al.,Proc. Natl. Acad. Sci. USA, 98, 7443-7448 2001). Combinations of twoanti-HGF/SF monoclonal antibodies still lacked full blocking activitywhile several mixtures of three monoclonal antibodies potentlyneutralized all HGF/SF activity in in vitro assays. It was concludedthat blocking of the biological activities of HGF/SF requires thesimultaneous binding of multiple monoclonal antibodies against differentepitopes of the HGF/SF ligand (B. Cao et. al., Proc. Natl. Acad. Sci.USA, 98, 7443-7448 2001).

Mixtures of monoclonal antibodies directed against the same targetmolecule that block the complete spectrum of biological activities ofthe molecule are very valuable contributions to the therapeutic arsenal,especially when such blocking activities can not be achieved withmonoclonal antibodies. Production of such multiple antibodies in apharmaceutical manner and in a commercially viable way will become veryimportant. In this example, we describe how mixtures of monoclonalantibodies against the HGF/SF ligand are isolated and used to constructan Oligoclonics™ that efficiently blocks all biological activities ofthis ligand.

Phage antibody scFv or Fab libraries that are formed by focusing thediversity in one variable region and keeping the other variable regioninvariable, preferably a germ line sequence, are particularly relevantto the invention. From such libraries it is feasible to isolateantibodies with a different heavy chain yet identical light chain, orvice versa (FIG. 3). Such antibodies are readily reformatted into anOligoclonics™ format according to the invention. In the art, it has beendescribed that antibodies that share the same VL gene but have differentVH genes and widely varying specificities can be obtained from phageantibody display libraries (Nissim et al. (1994), EMBO J. 13:692-698). Asub-library of the semi-synthetic scFv library (de Kruif et al. (1995)J. Mol. Biol. 248:97) described in Example 7 is used to selectantibodies against recombinant human HGF/SF.

The HGF/SF ligand is produced and purified from S-114 cells (NIH 3T3cells transformed with human HGF/SF and Met) as described (S. Rong etal. (1993) Cell Growth Differ. 4, 563-569). For phage selections,96-well plates are coated with 2.5 μg/ml HGF/SF in coating buffer (0.2 MNa₂CO₃/NaHCO₃, pH 9.6; 50 μl per well) overnight at 4° C. The plateswere then blocked with PBS containing 1% BSA (200 μl/well) overnight at4° C. Selections of phages binding to human HGF/SF are performed asdescribed in the previous examples. The binding of phages selected fromthe library is monitored by a HGF/SF ELISA using 96-well plates coatedwith 2.5 μg/ml HGF/SF in coating buffer (0.2 M Na₂CO₃/NaHCO₃, pH 9.6; 50μl per well) overnight at 4° C. The plates are then blocked with PBScontaining 1% BSA (200 μl/well) overnight at 4° C.

The VH regions from individual monoclonal antibodies and the single VLregion are cloned into the eukaryotic expression vector for humanmonoclonal antibodies as described in Example 10 and subsequentlyintroduced into eukaryotic CHO cells by transfection. For eachtransfection, the plasmids encoding two or more different VH regions aremixed in various ratios and used at a concentration of 1 to 10 μg/ml.Clones secreting human antibodies are generated essentially as describedin Example 10 and the supernatants monitored for HGF/SF-specificantibodies with an ELISA in 96-well plates coated with HGF/SF asdescribed in the previous paragraph. Supernatants from clones secretinganti-HGF/SF antibodies are used to determine the capacity of mixtures toblock the biological activities of HGF/SF.

Supernatants from transfectants are screened for neutralizing HGF/SFcapacity in the Madin-Darby canine kidney (MDCK) scatter assay asdescribed (B. Cao et. al., Proc. Natl. Acad. Sci. USA, 98, 7443-74482001). MDCK cells are plated at 7.5×10⁴ cells per 100 μl per well withor without HGF (5 μg/well) in DMEM with 5% FBS. Three hundredmicroliters of supernatants at two-fold serial dilutions is then addedto 96-well plates. A rabbit polyclonal-neutralizing antiserum (1μl/well; ref S. Koochekpour et. al. (1997) Cancer Res. 57, 5391-5398) isincluded as control. Following overnight incubation at 37° C., cells arethen stained with 0.5% crystal violet in 50% ethanol (vol/vol) for tenminutes at room temperature, and scattering is viewed using a lightmicroscope.

Supernatants from transfectants are also screened for neutralizingHGF/SF capacity in the Branching Morphogenesis Assay as described.Branching morphogenesis assay using SK-LMS-1 cells was conducted asdescribed (M. Jeffers et al. (1996) Mol. Cell Biol. 16, 1115-1125).Briefly, cell suspensions are mixed with an equal volume of GFR-Matrigel(Becton Dickinson), plated at 5×10⁴ cells per 125 μl per well in a96-well culture plate, and incubated for 30 minutes at 37° C. HGF/SF,with or without neutralizing mAbs, is added along with DMEM containing10% FBS on top of the gel. After 72 to 96 hours of incubation at 37° C.,representative wells are photographed at ×400 magnification.

Example 19 Pairing-Compatible Antibodies to Produce a Mixture ofAntibodies that Block Vascular Endothelial Cell Growth Factor Receptor 1(VEGF-R1) and VEGF-R2

Vascular endothelial growth factor (VEGF) is a key regulator ofangiogenic processes during adult life such as wound healing, diabeticretinopathy, rheumatoid arthritis, psoriasis, inflammatory disorders andtumor growth and metastasis (N. Ferrara et. al., Curr Top. Microbiol.Immunol. 237-1-30 (1999); M. Klagsbrun et al., Cytokine Rev. 7, 259-270(1996); G. Neufeld et al. FASEB J. 13, 9-22 (1999)). VEGF binds to andmediates its activity mainly through two tyrosine kinase receptors,VEGF-R1 (also named Flt-1) and VEGF-R-2 (also named KDR). Numerousstudies have shown that overexpression of VEGF and its receptors plays arole in associated-associated angiogenesis and hence in tumor growth andmetastasis (J. Folkman, J. Nat. Med. 1, 27-31 (1995); Z. Zhu et. al.,Invest. New Drugs 17, 195-212 (1999)).

A human anti-VEGF monoclonal antibody binding to VEGF and blocking itsbinding to the VEGF-R1 has recently been approved by the FDA for thetreatment of patients with metastatic colorectal cancer(http://www.fda.gov/cder/foi/appletter/2004/1250851tr.pdf). This showsthat monoclonal antibodies that block angiogenesis provide an importanttool in the treatment of solid tumors.

In WO/04003211A1, Zhu describes bispecific antibodies with one part ofthe molecule blocking the binding of VEGF to VEGF-R1 and another part ofthe molecule blocking binding of VEGF to VEGF-R2. In addition, thebi-specific antibody prevents the homodimerization of the VEGF receptorsand thus blocking VEGF-R-mediated cellular signaling. Compared tobinding to a single VEGF-R, dual binding can result in more potentinhibition of VEGF-stimulated cellular functions such as, for example,proliferation of endothelial cells. The bispecific antibodies describedby Zhu comprise single chain Fv antibody fragments fused to the heavyand light chain constant regions of an IgG molecule. Because of thenature of the bispecific molecules, they can be expected to beimmunogenic upon injection in humans, impeding their clinicaleffectiveness. Mixtures of human antibodies as represented in theOligoclonics™ format that block both the VEGF-R1 and VEGR-R2 whileretaining optimal clinical efficacy may be an important addition to thearsenal of anti-solid tumor drugs. Such an Oligoclonics™ is obtained asfollows:

The soluble fusion protein VEGF-R2 fused to alkaline phosphatase(VEGF-R2-AP) is expressed in stably-transfected NIH 3T3 cells andpurified from cell culture supernatant by affinity chromatography asdescribed (D. Lu et al., J. Biol. Chem. 275, 14321-14330 (2000)).VEGF-R1-Fc fusion protein is purchased from R&D Systems (Minneapolis,Minn.). VEGF-R2-AP is coated to Maxisorp Star tubes plates at aconcentration of 10 μg/ml and subsequently, the tubes are blocked with3% milk/PBS as described in WO 003211 and D. Lu et al., Cancer Res.61:7002-7008 (2001). The phage library used for selection of scFvantibody fragments specific for VEGF-R2 contains a single light chainand is diversified in the heavy chain as described in the previousExample 7. Selection of phages is carried out as described in theprevious examples. The specificity of selected scFv antibody fragmentsis determined in ELISA with 10 μg/ml VEGF-R2-AP coated to Maxisorp96-well plates and scFv binding, washing and detection steps asdescribed in the previous examples. As a control for binding to the APmoiety, scFv are assayed for binding to a control AP fusion proteinssuch as ELF2-AP (GenHunter Corp., Nashvffle, Tn). Selection of phagesbinding to the VEGF-R1 is carried out by coating Maxisorp Star tubeswith 10 μg/ml VEGF-R1-Fc and performing rounds of selection as describedin the previous examples. The specificity of selected scFv is analyzedin ELISA with 10 μg/ml VEGF-R1-Fc coated to 96-well plates. As a controlfor binding to the Fc portion VEGF-R1-Fc, plates are coated with the Fcfusion protein rhsThy-1:Fc (product number ALX-203-004, AlexisBiochemicals, Lausen, Switzerland).

The VH regions from individual monoclonal antibody fragments and thesingle VL region are cloned into the eukaryotic expression vector forhuman monoclonal antibodies as described in Example 10 and subsequentlyintroduced into eukaryotic CHO cells by transfection. For eachtransfection, the plasmids encoding two or more different VH regions aremixed in various ratios and used at a concentration of 1 to 10 μg/ml.Clones-secreting human antibodies are generated essentially as describedin Example 10 and the supernatants monitored for VEGF-R1 andVEGF-R2-specific antibodies with an ELISA in 96-well plates coated withVEGF-R1-Fc and VEGF-R2-AP as described in the previous paragraph, andusing secondary antibodies that specifically bind to the humanantibodies. Supernatants from clones secreting antibodies to bothreceptors are used to determine the biological activity of the mixturesin VEGF-R1 and VEGF-R2 blocking assays and in an anti-mitotic andleukemia migration assays.

VEGF-R1 and VEGF-R2 blocking assays are performed as described (Z. Zhuet al., Cancer Res. 58:3209-14 (1998); D. Lu et al., J. Immunol.Methods, 230:159-71 (1999). The anti-mitotic and leukemia migrationassays are performed as described in WO 04003211A1. To measure whetherthese antibody mixtures compete with VEGF for binding to the receptors,assays are carried out that measure the level of antibody-inducedinhibition of VEGF-associated effects. For example, the effect of theantibody cocktail on VEGF-induced endothelial cell proliferation ismeasured using a thymidine incorporation assay. Numerous in vitro and invivo assays have been described to measure the effect of ligandsinterfering with the VEGF-VEGF-receptor interaction. Some suitableassays are described in Gerbert et al., J. Biol. Chem. 1998, 273:30336(cell survival assay, endothelial cell apoptosis, Akt phosphorylationassay, as on page 30337); in Mendel et al., Clin. Cancer Res. 2000,6:4848-4858 (s.c. xenograft model in athymic mice, surface expression ofKDR, ¹²⁵I VEGF binding assay, and Flk-1 receptor kinase assay, as onpages 4849-4850). These and other suitable assays are reviewed inAuerbach et al., 2003, Clin. Chemistry 49(1):32-40.

1. A method for selecting a single recombinant cell that expresses aheterogeneous combination of monospecific and bispecific antibodies, orantibody fragments thereof, wherein the monospecific and bispecificantibodies, or antibody fragments thereof, are human, humanized, ordeimmunized, and wherein the heterogeneous combination has specificaffinity for two target epitopes, said method comprising: carrying out aprocess for producing the heterogeneous combination; the processcomprising providing three different variable regions consisting of twoheavy chain regions and one light chain variable region in recombinantcells, wherein antigen binding parts of the variable regions originatefrom an antibody from a single species and wherein one variable regionis able to functionally pair with more than one other variable region,and under conditions allowing for pairing of variable regions andsecretion of the paired regions from the recombinant cells resulting inthe production of said heterogeneous combination, and providing twotarget epitopes, selecting a single recombinant cell from saidrecombinant cells that produces a heterogeneous combination that bindsthe two target epitopes, and co-purifying the heterogeneous combinationtherefrom utilizing a shared feature of each of the monospecific andbispecific antibodies, or antibody fragments thereof.
 2. The methodaccording to claim 1, wherein said two target epitopes are associatedwith a disease and/or disorder.
 3. The method according to claim 2,further comprising: subjecting a heterogeneous combination to abiological assay indicative of an effect of the combination on thedisease and/or disorder.
 4. The method according to claim 1, wherein theone variable region able to functionally pair with more than one othervariable region does not significantly contribute to the resultingbinding specificity of the resulting paired regions.
 5. The methodaccording to claim 1, wherein each variable region can only pair withone other variable region.
 6. The method according to claim 1, whereintwo of the three variable regions are part of one single chain Fv. 7.The method according to claim 1, wherein the expression of two of thevariable regions is under the direction of different control elements.8. The method according to claim 7, wherein the different controlelements lead to differential expression.
 9. The method according toclaim 8, wherein the differential expression is different in levels ofexpression and/or time of expression.
 10. The method according to claim1, wherein the combination comprises two monospecific antibodiesproduced in the single recombinant cell.
 11. The method according toclaim 1, further comprising producing an expression system comprisingnucleic acid sequences encoding variable regions; said producingcomprising: synthesizing nucleic acid sequences encoding variableregions, expressing said nucleic acid sequences and allowing theexpression products to pair, and selecting nucleic acid sequencesencoding variable regions having desired pairing behavior, so as toproduce nucleic acid sequences encoding variable regions.
 12. A methodfor producing a heterogeneous combination of monospecific and bispecificantibodies, or antibody fragments thereof, the method comprising:carrying out a selection method according to claim 1, and allowing theselected cell to express the heterogeneous combination of monospecificand bispecific antibodies, or antibody fragments thereof, resulting inthe production of said heterogeneous combination.
 13. The methodaccording to claim 1, wherein the shared feature is an Fc region.